Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Molecular characterization of a precision-cut rat liver slice model for the evaluation of antifibrotic compounds

doi: 10.1152/ajpgi.00281.2018

Figure Lengend Snippet: ELISA analysis of secreted biomarkers. SB525334, nintedanib, and sorafenib suppressed secretion of hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) in the conditioned medium after 48 h of incubation. Data are means ± SE; n = 3 rats for both sham and bile duct ligation (BDL); n = 8–10 liver slices for each condition. **P < 0.01 and ***P < 0.001.

Article Snippet: Secreted biomarkers including procollagen I, HA, IGFBP5, and WISP1 in the conditioned medium were measured using procollagen I ( {"type":"entrez-nucleotide","attrs":{"text":"Ab210579","term_id":"70570352","term_text":"AB210579"}} Ab210579 ; Abcam, Cambridge, UK), hyaluronan quantikine (DHYAL0; R&D Systems, Minneapolis, MN), IGFBP5 ( {"type":"entrez-nucleotide","attrs":{"text":"Ab208345","term_id":"76667415","term_text":"AB208345"}} Ab208345 ; Abcam), and WISP1 quantikine (MWSP10; R&D Systems) ELISA kits following manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Ligation

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Molecular characterization of a precision-cut rat liver slice model for the evaluation of antifibrotic compounds

doi: 10.1152/ajpgi.00281.2018

Figure Lengend Snippet: The small molecule αV integrins inhibitor CWHM12 attenuated fibrotic phenotype in fibrotic precision-cut liver tissue slices (PCLSs) after 48 h of incubation. A: CWHM12 suppressed expression of a panel of fibrotic genes. B: CWHM12 decreased secretion of procollagen I, hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) in the conditioned medium. Data are means ± SE; n = 3 rats for both sham and bile duct ligation (BDL); n = 6–8 liver slices for each condition. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Secreted biomarkers including procollagen I, HA, IGFBP5, and WISP1 in the conditioned medium were measured using procollagen I ( {"type":"entrez-nucleotide","attrs":{"text":"Ab210579","term_id":"70570352","term_text":"AB210579"}} Ab210579 ; Abcam, Cambridge, UK), hyaluronan quantikine (DHYAL0; R&D Systems, Minneapolis, MN), IGFBP5 ( {"type":"entrez-nucleotide","attrs":{"text":"Ab208345","term_id":"76667415","term_text":"AB208345"}} Ab208345 ; Abcam), and WISP1 quantikine (MWSP10; R&D Systems) ELISA kits following manufacturer's instructions.

Techniques: Incubation, Expressing, Binding Assay, Ligation

Journal: The Journal of Neuroscience

Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend Snippet: TSP-receptor α2δ-1 is synaptically expressed in the retina, and its expression is reduced in RCS rats. A, Representative images of the retina stained for α2δ-1 from LE (healthy) and RCS (degenerative) retinas at P14 and (B) P30. C, Quantitative staining intensity analysis demonstrated that α2δ-1 expression was reduced in RCS rat as early as P14. D, α2δ-1 was enriched in both the OPL and IPL, and the expression gap became more distinct at P30. Representative images of the (E) OPL and (F and G) IPL with the synapses labeled for Bassoon (F, green), VGluT1 (G, green), α2δ-1 (red), and NR1 (blue) from LE retina on P21 demonstrated postsynaptic expression of α2δ-1. Representative images of the (H) OPL and (J) IPL synapses labeled for Bassoon (green) and α2δ-1 (red) from LE (healthy) and RCS (degenerative) retinas on P21. Quantification of α2δ-1-containing synapses formed in the (I) OPL and (K) IPL reveals that the number of α2δ-1 synapse is already reduced in RCS rat by P21. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. ***p < 0.0001.

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: AB_10618753 , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: AB_87705 , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: AB_1279448 , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: AB_2201958 , AF3074, R&D Systems], goat-anti-TSP2 1:200 [RRID: AB_2202068 , AF1635, R&D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: AB_397879 , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: AB_2239761 , AB5535, Millipore], goat anti-ChAT[RRID: AB_11214092 , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: AB_258885 , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: AB_2039785 , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: AB_10013382 , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: Expressing, Staining, Labeling

Journal: The Journal of Neuroscience

Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend Snippet: Subretinal hUTC transplantation preserves OPL synapses in RCS rats. Representative images of the OPL with the PR ribbon synapses labeled for (A) Bassoon (green) and mGluR6 (red) and (B) Bassoon (green) and α2δ-1 (red) from LE (control), RCS + BSS, and RCS + hUTC P21 and P60 retinas on P95. Quantification of the number of synapses in the OPL revealed that hUTC transplantation protected (C) ribbon synapses. D, Particularly, α2δ-1-containing synapses were specifically preserved following hUTC treatment. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. ***p < 0.0001.

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: AB_10618753 , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: AB_87705 , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: AB_1279448 , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: AB_2201958 , AF3074, R&D Systems], goat-anti-TSP2 1:200 [RRID: AB_2202068 , AF1635, R&D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: AB_397879 , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: AB_2239761 , AB5535, Millipore], goat anti-ChAT[RRID: AB_11214092 , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: AB_258885 , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: AB_2039785 , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: AB_10013382 , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: Transplantation Assay, Labeling

Journal: The Journal of Neuroscience

Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend Snippet: Subretinal hUTC transplantation preserves α2δ-1-containing synapses in the IPL of RCS rats. A, Representative images of the IPL labeled for VGluT1 (green) and PSD95 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. B, Quantification of excitatory synapses in the IPL revealed that synapse numbers did not differ between RCS+BSS and RCS+hUTC P21 and P60. C, Representative images of the IPL labeled for Bassoon (green) and α2δ-1 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. D, The α2δ-1-containing synapses were specifically preserved with hUTC transplantation. E, Representative images of the IPL stained for Bassoon (green) and Gephyrin (blue) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. F, hUTC transplantation did not preserve inhibitory synapses. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *p < 0.05.

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: AB_10618753 , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: AB_87705 , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: AB_1279448 , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: AB_2201958 , AF3074, R&D Systems], goat-anti-TSP2 1:200 [RRID: AB_2202068 , AF1635, R&D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: AB_397879 , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: AB_2239761 , AB5535, Millipore], goat anti-ChAT[RRID: AB_11214092 , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: AB_258885 , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: AB_2039785 , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: AB_10013382 , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: Transplantation Assay, Labeling, Staining

Journal: The Journal of Neuroscience

Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend Snippet: KO of MERTK in MG results in impaired synapse development in vivo. A, Representative images of the OPL synapses labeled by Bassoon (green) and mGluR6 (red) from Control (CTR, right eyeballs) and MERTK KO (KO, left eyeballs) at P21 and P45. B, Quantification of synapses in the OPL revealed that synapse development is impaired in MG-specific MERTK-KO at both P21 and P45. C, IPL excitatory synapses are visualized by VGluT1 (green) and PSD95 (red). D, Quantification of synapse number demonstrates that early synaptic development at P21 is significantly affected by MERTK KO but not at P45. Representative images of the (E) OPL and (G) IPL synapses labeled for Bassoon (green) and α2δ-1 (red). Quantification of the number of α2δ-1-containing synapses in both (F) OPL and (H) IPL revealed that KO of MERTK in MG induces severely reduced number of α2δ-1-containing synapses at P21 and P45. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *p < 0.05.

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: AB_10618753 , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: AB_87705 , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: AB_1279448 , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: AB_2201958 , AF3074, R&D Systems], goat-anti-TSP2 1:200 [RRID: AB_2202068 , AF1635, R&D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: AB_397879 , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: AB_2239761 , AB5535, Millipore], goat anti-ChAT[RRID: AB_11214092 , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: AB_258885 , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: AB_2039785 , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: AB_10013382 , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: In Vivo, Labeling

Journal: American Journal of Physiology - Renal Physiology

Article Title: Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome

doi: 10.1152/ajprenal.00207.2017

Figure Lengend Snippet: ADR-induced injury induced and activated small heat shock proteins (HSPs), primarily in an MK2-dependent manner. Total protein was extracted from renal cortexes isolated from control and ADR-injured mice on day 21 from each genotype and phosphorylated and total forms of HSPB1, HSPB8, glucose-regulated protein 78 (GRP78), and GAPDH were detected using their respective antibodies. A: representative Western blots of total and phosphorylated forms of selected proteins. B: densitometry analyses of protein induction/phosphorylation in the renal cortexes of at least 3 mice from each genotype. Statistical differences: *P < 0.05, control vs. ADR; #P < 0.05, WT-ADR treated vs. KO-ADR treated.

Article Snippet: The other primary antibodies used were anti-HSPB1 (StressMarq, Victoria, BC), anti-HSPB8 mouse monoclonal (Abcam, Cambridge, MA), anti-GRP78 rabbit monoclonal (StressMarq), and anti-GAPDH mouse monoclonal (Millipore, Billerica, MA).

Techniques: Isolation, Western Blot