Journal: Journal of Cellular and Molecular Medicine

Article Title: Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling

doi: 10.1111/jcmm.12230

Figure Lengend Snippet: UII inhibits the phosphorylation of LRP6 in cardiac side population cells (CSPs) by UT/JNK signaling during mechanical stretch. ( A ) Immunofluorescent images revealed that positive signal (green) could be observed in cultured CSPs with antibody to p-LRP6 (1:200); bar: 20 μm. ( B ) Quantitative evaluation of p-LRP6 by analysis of fluorescence intensity. ( C ) P-LRP6 was detected in cultured CSPs by nanofluidic proteomic immunoassay (NIA). Peaks on the traces that represent phosphorylated isoforms of LRP6 are indicated. ( D ) NIA pseudoblot representation of p-LRP6 and bar graph of p-LRP6/GAPDH in cultured CSPs by NIA quantification. Cultured CSPs were pre-treated with PBS, urantide (Ura, 1 μM) or SP600125 (SP6, 5 μM), respectively, for 30 min, then subjected to mechanical stretch (MS) and incubated with PBS or UII (0.1 μM) for 3 hrs. Values are expressed as mean ± SEM. ** P < 0.01 versus control; ## P < 0.01 versus control; # P < 0.05 versus MS; & P < 0.05 versus MS plus UII; && P < 0.01 versus MS plus UII. The experiment was repeated for at least three times. ( E ) P-LRP6 was detected in CSPs isolated from transverse aorta constriction (TAC) or Sham mice by NIA. ( F ) NIA pseudoblot representation of p-LRP6 and bar graph of p-LRP6/GAPDH in isolated CSPs by NIA quantification. Values are expressed as mean ± SEM. Sham: n = 9; Sham+Ura: n = 6; TAC: n = 6; TAC+Ura: n = 7. Ura: urantide. ** P < 0.01 versus Sham mice; ## P < 0.01 versus TAC mice.

Article Snippet: Lipoprotein receptor related protein 6 (LRP6) -pCS2 (Addgene, from Dr. Xi He) was transfected to UT cells to induce LRP6 overexperssion.

Techniques: Phospho-proteomics, Cell Culture, Fluorescence, Incubation, Control, Isolation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling

doi: 10.1111/jcmm.12230

Figure Lengend Snippet: UII inhibits the phosphorylation of LRP6 in UT cell line by activating JNK during mechanical stretch. Western blot analysis for p-LRP6 and p-JNK levels. UT cells were pre-treated with Urantide (Ura, 1 μM) for 30 min., then subjected to MS and treated with UII for 30 min. and 3 hrs. ( A ) Representative picture of p-LRP6 and p-JNK level at 30 min. after UII treatment during MS. Quantitative evaluation of p-JNK ( B ) and p-LRP6 ( C ) by analysis of Western blot. ( D ) Representative picture of p-LRP6 and p-JNK level at 3 hrs after UII treatment during MS. Quantitative evaluation of p-JNK ( E ) and p-LRP6 ( F ) by analysis of Western blot. P-JNK and p-LRP6 were analysed in UT cells pre-treated with SP600125 (SP6, 5 μM; G ) or overexpressed LRP6 ( H ) with or without UII (0.1 μM) during MS. UT cells were pre-treated with SP600125 or DMSO for 30 min., then subjected to MS and treated with UII for 3 hrs. UT cells were overexpressed with LRP6-pCS2 or pCS2 for 48 hrs, then subjected to MS and treated with UII for 3 hrs. Values are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 versus control. # P < 0.05, ## P < 0.01 versus MS. & P < 0.05, && P < 0.01 versus MS plus UII. The experiment was repeated for at least three times.

Article Snippet: Lipoprotein receptor related protein 6 (LRP6) -pCS2 (Addgene, from Dr. Xi He) was transfected to UT cells to induce LRP6 overexperssion.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling

doi: 10.1111/jcmm.12230

Figure Lengend Snippet: UII inhibits the proliferation of cardiac side population cells (CSPs) by c-Jun N -terminal kinase (JNK)-LRP6 signalling during mechanical stretch. CSPs proliferation was determined by Cell Viability Assay. Cultured CSPs were overexpressed LRP6 ( A ) or pretreated with SP600125 (SP6, 5 μM; B ) with or without UII (0.1 μM) during mechanical stretch (MS). CSPs were pre-treated with SP600125 or DMSO for 30 min., then subjected to MS and treated with UII for 48 hrs. CSPs were overexpressed with LRP6-pCS2 or pCS2 for 48 hrs, then subjected to MS and treated with UII for 48 hrs. Values are expressed as mean ± SEM. ** P < 0.01 versus MS. # P < 0.05 versus MS plus UII. The experiment was repeated for at least three times. ( C ) The ratio of CSPs per mouse was analysed by fluorescence-activated cell sorting (FACS). Representative photographs are shown. ( D ) The ratio of CSPs per mouse was calculated by FACS. SP600125 (SP6) or DMSO were injected intraperitoneally either with 40 mg/kg bodyweight every 3 days for 2 weeks. At 4 weeks after transverse aorta constriction (TAC) or sham operation, the number of CSPs was determined. Values are expressed as mean ± SEM. Sham: n = 5; Sham+SP6: n = 6; TAC: n = 6; TAC+SP6: n = 6. Ura: urantide. * P < 0.05 versus sham mice; # P < 0.05 versus TAC mice.

Article Snippet: Lipoprotein receptor related protein 6 (LRP6) -pCS2 (Addgene, from Dr. Xi He) was transfected to UT cells to induce LRP6 overexperssion.

Techniques: Viability Assay, Cell Culture, Fluorescence, FACS, Injection

Journal: iScience

Article Title: Disuse plasticity limits spinal cord injury recovery

doi: 10.1016/j.isci.2025.112180

Figure Lengend Snippet: Early disuse persists synaptic expression of GluA2-lacking AMPARs as changes in synapse numbers on ventral horn neurons and dendritic neuropils after spinal cord injury Overdrive of glutamate AMPARs is known to reflect maladaptive spinal cord plasticity in central nervous system trauma. , (A–E) Workflow diagram of the automated and unbiased high-resolution robotic confocal microscopy. To assess synaptic levels of glutamate AMPARs on dendritic fields and somata of large ventral horn neurons, the randomized microscopic detection and analysis were performed blindly by spinning disk confocal scan system at week 8 post-injury according to the established algorithm. , (A) Fluorescently labeled large ventral horn neurons (diameter >40 μm) indicated presumptive motorneurons and were detected centrally in the sampling window (80 × 80 μm) at 63× magnification. (B) A stack of high-resolution images was taken in the z-plane through a 650 nm filter for presynaptic synaptophysin (upper panel) and a 490 nm filter for postsynaptic AMPARs (lower panel) at each level separately (scale bar: 20 μm). (C) Scanned confocal z stacks were deblurred by 3-D blind iterative deconvolution, and then the total expression of synaptic colocalized AMPAR puncta was quantified using the established approach. (D) Single optical planes showing maximal synaptic colocalization of presynaptic synaptophysin and postsynaptic AMPARs on the somata were selected among the z stacks, and then the optical fraction images of the somatic membrane were generated to assess the synaptic AMPAR expression on the plasma membrane (left panel). Representative optical detection of the presynaptic vesicle, postsynaptic AMPAR subunit, and synaptic colocalization (white arrows) are shown in the enlarged image of the boxed region from the plasma membrane image (right panel; see Figure 5 ). (E) Schematic overview on synaptic colocalization of AMPAR subunits GluA1 and GluA2. The overlapping of presynaptic synaptophysin (magenta) and synaptic AMPAR subunit (GluA1 or GluA2; green) puncta indicates synaptic colocalization (white). (F) Representative merged 3-D confocal image of large ventral horn neurons showed postsynaptic GluA1 (green) and synaptophysin (magenta) positive presynaptic terminals on the soma with surrounding dendritic neuropils in the control subject (left panel). The enlarged image of the boxed region from (F, left panel) demonstrates relatively few synaptic colocalized GluA1 puncta (lower right panel, white) within the merge image (upper right panel) in the control subject. (G) Representative merged motorneuron from the early disuse subject demonstrating greater numbers of membrane and dendritic GluA1 colocalized to the synapses (left panel). The enlargement of the boxed region from (G, left panel) demonstrates high levels of synaptic colocalized GluA1 puncta (lower right panel, white) within the merge image (upper right panel) in the early disuse subject. Representative images of synaptic GluA2 puncta are shown in Figure S8 . (H) Quantification of synaptic colocalized AMPAR expression with synaptophysin though the confocal z stacks in the early disuse group ( n = 5; total 164 cells, total 11,971 optical planes for GluA1; total 138 cells, total 9,788 planes for GluA2) compared to the control group ( n = 6; total 163 cells, total 13,037 images for GluA1; total 156 cells, total 10,894 optical planes for GluA2) on the somata and dendritic neuropil. Random effects ANOVA controlling for non-independence of within-subject and within-section variability confirmed that early disuse significantly increased in synaptic colocalization of GluA1 (effect of early disuse condition: F (1,9) = 8.05, ∗ p = 0.019) but no significant difference in synaptic colocalization of GluA2 (effect of early disuse condition: F (1,9) = 0.074, p = 0.792). There was a non-significantly difference between both groups in the presynaptic synaptophysin and postsynaptic AMPAR subunit ( Figure S8 ). Representative examples reflect the group median. ∗ p < 0.05 by one-way ANOVA. All data are shown as means ± SEM. Scale bars in the lower magnification images represent 20 μm, and the scale bar in the higher magnification image represents 5 μm.

Article Snippet: Slides were washed repeatedly with PBS and then coverslipped with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). (2) Confocal image acquisition and 3-D blind iterative image deconvolution All images were acquired at the UCSF Nikon Imaging Center using a Ti inverted microscope (Nikon Instruments, Melville, NY) with a spinning disk confocal scan system (CSU-22; Yokogawa Corporation, Sugar Land, TX) and a Plan Apo VC 63× oil immersion objective with 1.4 numerical aperture (Nikon Instruments), electron multiplying CCD camera running a custom instance of NIS elements imaging software (Nikon Instruments).

Techniques: Expressing, Confocal Microscopy, Labeling, Sampling, Membrane, Generated, Clinical Proteomics, Control

Journal: iScience

Article Title: Disuse plasticity limits spinal cord injury recovery

doi: 10.1016/j.isci.2025.112180

Figure Lengend Snippet:

Article Snippet: Slides were washed repeatedly with PBS and then coverslipped with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). (2) Confocal image acquisition and 3-D blind iterative image deconvolution All images were acquired at the UCSF Nikon Imaging Center using a Ti inverted microscope (Nikon Instruments, Melville, NY) with a spinning disk confocal scan system (CSU-22; Yokogawa Corporation, Sugar Land, TX) and a Plan Apo VC 63× oil immersion objective with 1.4 numerical aperture (Nikon Instruments), electron multiplying CCD camera running a custom instance of NIS elements imaging software (Nikon Instruments).

Techniques: Recombinant, Protease Inhibitor, Blocking Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Software, Imaging, Inverted Microscopy, Microscopy