Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal weight (A) and umbilical artery serum DHAS (B) and cortisol (C) levels in untreated baboons on days 100 (middle, n = 4) and 165 (late, n = 8) of gestation and on day 165 in animals that were treated daily on days 100–164 with letrozole (0.115 mg/kg body weight per day, n = 8) or letrozole plus estradiol (each at 0.115 mg/kg body weight per day, n = 5). Values indicated by different letter superscripts are different at P < .05 to P < .01 (ANOVA and Newman-Keul's multiple comparison test).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques:

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Western immunoblot of MC2R (A) and MRAP (B) incubated with primary antibody (Ab), with primary Ab preabsorbed with blocking peptide or without primary Ab in the baboon fetal adrenal gland on day 165 of gestation in untreated and letrozole-treated baboons (n = 2 samples each). Panels C and D show Western immunoblots of MC2R and MRAP in the fetal liver and epididymis on day 165 in an untreated baboon.

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Western Blot, Incubation, Blocking Assay

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal MC2R protein expression assessed by PLA in the DZ/TZ (A and B) and FZ (C and D) on day 165 of gestation in baboons untreated (A and C) and treated with letrozole on days 100–164 (B and D). Panel E shows the means (±SE) of fetal adrenal MC2R protein expression, quantified by PLA and image analysis/Metamorph software, on day 100 (midgestation, n = 4) and day 165 (late gestation, n = 8) in untreated baboons and on day 165 in animals treated with letrozole (n = 8) or letrozole plus estradiol (n = 5). Each red PLA signal represents a single molecule of MC2R protein detected by primary MC2R antibody tagged with a secondary antibody conjugated to fluorescently labeled oligonucleotide. Nuclei are labeled blue. P-450C17 immunostaining with Alexa Fluor 488-conjugated IgG within TZ (A and B) and FZ (C and D) cells is shown in green. Final magnification, ×400 (A–D).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Expressing, Software, Labeling, Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal MRAP protein expression assessed by PLA in the DZ/TZ (A and B) and FZ (C and D) on day 165 of gestation in baboons untreated (A and C) and treated with letrozole (B and D). Panel E shows the means (±SE) of fetal adrenal MRAP quantified by PLA and image analysis/Metamorph software in the same baboons in which MC2R is shown in Figure 3. Nuclei are labeled blue. P-450C17 immunostaining within TZ (A and B) and FZ (C and D) are shown in green. Final magnification, ×400 (A–D). *, Significantly different at P < .03 in late vs midgestation.

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Expressing, Software, Labeling, Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal adrenal MC2R-MRAP protein interaction assessed by PLA in the DZ/TZ (A and B) and FZ (C and D) on day 165 of gestation in baboons untreated (A and C) and treated with letrozole (B and D). Panel E shows the means (±SE) of fetal adrenal MC2R-MRAP interaction quantified by PLA and image analysis/Metamorph software in the same baboons in which MC2R is shown in Figure 3. For PLA detection of MC2R-MRAP protein interaction, tissue was incubated with a secondary antirabbit PLUS antibody and a secondary antigoat minus antibody conjugated with oligonucleotide. Nuclei are labeled blue. P-450C17 immunostaining within TZ (A and B) and FZ (C and D) are shown in green. Final magnification, ×400 (A–D). *, Significantly different at P < .05 in letrozole-treated animals vs all other groups (ANOVA and Tukey-Kramer multiple comparison test).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Software, Incubation, Labeling, Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: Fetal liver hepatocyte (A and B) and fetal epidydimal stroma (D and E) MC2R-MRAP interaction assessed by PLA on day 165 of gestation in untreated (A and D) and letrozole-treated (B and E) baboons. α-Smooth muscle actin immunostaining within fibromuscular cells of epidydimal ducts appears in green in panels D and E. Means ± SE of fetal liver (C) and fetal epidydimal stroma (F) MC2R-MRAP interaction were quantified by PLA in baboons untreated (n = 3) or treated with letrozole (n = 3). Final magnification, ×400 in each panel.

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Immunostaining

Journal: Endocrinology

Article Title: Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex

doi: 10.1210/en.2016-1562

Figure Lengend Snippet: A, Fetal pituitary POMC mRNA expression was quantified by in situ hybridization on days 100 (n = 3) and 165 (n = 7) in untreated baboons and on day 165 in animals treated with letrozole (n = 7) or letrozole plus estradiol (n = 7). B, Fetal (ie, umbilical artery) plasma ACTH levels on days 100 (n = 8) and 165 (n = 18) in untreated baboons and on day 165 in animals treated on days 100–164 with letrozole (n = 14) or letrozole plus estradiol (n = 11). Fetal pituitaries for POMC mRNA assay and blood samples for plasma ACTH assay were obtained from animals of the current study and from a contemporaneous group of baboons from our primate colony. Values indicated by different letter superscripts are different at P < .05 (ANOVA and Newman-Keul's multiple comparison test).

Article Snippet: MC2R and MRAP Western immunoblot Western immunoblot analysis with goat anti-MC2R Santa Cruz Biotechnology C-16/6876 antibody demonstrated two distinct bands in extracts of whole fetal adrenal glands from untreated and letrozole-treated baboons ( A), an unmodified form at the predicted molecular weight of 34 kDa and a larger 45-kDa presumably N-glycosylated form of the receptor, consistent with that observed in c-Myc-hMC2R transfected M3 cells using a mouse anti-Myc antibody ( 41 ) and in 35 S-methionine labeled mouse YI adrenocortical cells immunoprecipitated with the Santa Cruz Biotechnology C-16 anti-MC2R antibody and a rabbit Santa Cruz Biotechnology anti-MC2R H-70 antibody ( 42 ).

Techniques: Expressing, In Situ Hybridization

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 4. The identification of YAP-pSmad2/3-SOX9 axis during MSCs chondrogenesis. (A) RNA-seq analyses in human SMSCs after static and CTS treatment [(A1) volcano plot of differentially expressed genes (DEGs) in CTS group versus static group; (A2) KEGG pathway enrichment, red boxes represent Hippo and TGFβ signaling]. (B) Volcano plot of differentially expressed genes in regenerated tissue of PCL scaffold + synovium transplant group versus native porcine menisci [(B1) PCL scaffold + synovium transplant group 2M versus native menisci of sham group; (B2) PCL scaffold + synovium transplant group 4M versus native menisci of sham group; (B3) PCL scaffold + synovium transplant group at 2 versus 4 months]. (C) Heatmap of differential gene cluster in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(C1) PCL scaffold + synovium transplant group 2M versus native menisci of sham group; (C2) PCL scaffold + synovium transplant group 4M versus native menisci of sham group; (C3) PCL scaffold + synovium transplant group at 2 versus 4 months]. (D) KEGG pathway enrichment in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(D1) PCL scaffold + synovium transplant group 2M versus native menisci of sham group; (D2) PCL scaffold + synovium transplant group 4M versus native menisci of sham group; (D3) PCL scaffold + synovium transplant group at 2 versus 4 months, the red boxes represent Hippo and TGFβ signaling]. (E) Protein and mRNA levels in human SMSCs after static and CTS treatment [(E1) Western blot analysis; (E2) semiquantitative analysis of Western blot, n = 3, unpaired t test; (E3) mRNA levels, n = 8, unpaired t test]. (F) Cell immunofluorescence of human SMSCs after static and CTS treatment [(F1) cell immunofluorescence; (F2) six regions are randomly selected for semiquantitative analysis; the intensity of immunofluorescence is calculated by ImageJ software, n = 6, unpaired t test] (continued with Fig. 5). **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: RNA Sequencing, Western Blot, Immunofluorescence, Software

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 5. The identification of YAP-pSmad2/3-SOX9 axis during MSCs chondrogenesis. (A) Protein and mRNA levels in human SMSCs after being treated with dihy- drexidine and SIS3 [(A1) the Western blot analysis; (A2) the semiquantitative analysis of Western blot, n = 3, one-way ANOVA; (A3) the alcian blue staining; (A4) the semiquantitative analysis of alcian blue staining, n = 3, one-way ANOVA; (A5) cell immunofluorescence; (A6) the semiquantitative analysis of cell immunofluorescence, Dihy represents dihydrexidine, n = 6, one-way ANOVA; (A7) the mRNA levels, n = 8, one-way ANOVA]. (B) Immunofluorescent colocalization of YAP and Smad2/3 in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(B1) immunofluorescent colocalization of YAP and Smad2/3; (B2) immu- nofluorescence overlap coefficient, n = 9]. (C) Immunofluorescent colocalization of pSmad2/3 and SOX9 in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(C1) immunofluorescent colocalization of pSmad2/3 and SOX9; (C2) immunofluorescence overlap coefficient, n = 9]. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Western Blot, Staining, Immunofluorescence

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 6. Piezo1 mediates mechanotransduction on YAP-pSmad2/3-SOX9 axis through concerted activation of calcineurin and NFATc1 during MSCs chondro- genesis. (A) Protein and mRNA levels of Piezo1 in human SMSCs after static and CTS treatment [(A1) the Western blot analysis; (A2) the semiquantitative analysis of Western blot, n = 3, unpaired t test; (A3) the cell immunofluorescence; (A4) the semiquantitative analysis of cell immunofluorescence, n = 6, unpaired t test; (A5) the mRNA levels, n = 8, unpaired t test]. (B) Immunofluorescent assessment of Piezo1 expression in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci. (C) Protein and mRNA levels in human SMSCs after being treated with YODA1 or GsMTx-4 [(C1) the Western blot analysis; (C2) the semiquantitative analysis of Western blot, n = 3, one-way ANOVA; (C3) the mRNA levels, n = 8, unpaired t test; (C4) the alcian blue staining; (C5) the semiquantitative analysis of alcian blue staining, n = 3, one-way ANOVA; (C6) the cell immunofluorescence; (C7) the semiquantitative analysis of cell immunofluorescence, n = 6, one-way ANOVA]. (D) Assessment of intracellular calcium content in human SMSCs [(D1) intracellular calcium content after static and CTS treatment; (D2) the semiquantitative analysis of calcium content, n = 5, unpaired t test; (D3) intracellular calcium content after being treated with YODA1 or GsMTx-4; (D4) the semiquantitative analysis of calcium content, n = 5, one-way ANOVA]. (E) Protein and mRNA levels in human SMSCs after CsA treatment [(E1) Western blot analysis, the red arrow represents dephosphorylated NFATc1, the blue arrows represent phosphorylated NFATc1; (E2) the semiquantitative analysis of Western blot, n = 3, one-way ANOVA; (E3) the alcian blue staining; (E4) the semiquantitative analysis of alcian blue staining, n = 3, one-way ANOVA; (E5) the mRNA levels, n = 8, unpaired t test; (E6) the cell immunofluorescence; (E7) the semiquantitative analysis of cell immunofluorescence, n = 6, one-way ANOVA]. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Activation Assay, Western Blot, Immunofluorescence, Expressing, Staining

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 7. The effect of matrix stiffness on YAP-pSmad2/3-SOX9 axis. (A) Protein and mRNA levels in human SMSCs after being treated with soft or stiff matrix [(A1) the mechanical properties of silicon rubber and polystyrene culture plate, n = 3; (A2) Western blot analysis, the red arrow indicates dephosphorylated NFATc1, the blue arrows indicate phosphorylated NFATc1; (A3) the semiquantitative analysis of Western blot, n = 3, unpaired t test; (A4) the alcian blue staining; (A5) the semiquantitative analysis of alcian blue staining, n = 3, unpaired t test; (A6) the mRNA levels, n = 8, unpaired t test]. (B) Immunofluorescent assessment of FAK in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(B1) the immunofluorescence of FAK; (B2) the semiquantitative analysis of FAK, n = 6, one-way ANOVA]. (C) Immunofluorescent assessment of pFAK in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci [(C1) the immunofluorescence of pFAK; (C2) the semiquantitative analysis of pFAK, n = 6, one-way ANOVA]. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Western Blot, Staining, Immunofluorescence

Journal: Science advances

Article Title: Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

doi: 10.1126/sciadv.adg8138

Figure Lengend Snippet: Fig. 8. The biomechanical stimulus and matrix stiffness regulate the expression of collagen cross-link enzymes (LOX and LH) through Piezo1. (A) Protein and mRNA levels of LOX and LH in human SMSCs after static or CTS treatment [(A1) the Western blot analysis; (A2) the semiquantitative analysis of LOX, n = 3, unpaired t test; (A3) the semiquantitative analysis of LH2, n = 3, unpaired t test; (A4) the cell immunofluorescence of LOX; (A5) the semiquantitative analysis of LOX immunofluorescence, n = 6, unpaired t test; (A6) the cell immunofluorescence of LH2; (A7) the semiquantitative analysis of LH2 immunofluorescence, n = 6, unpaired t test; (A8) the mRNA levels, n = 8, unpaired t test]. (B) Protein and mRNA levels of LOX and LH in human SMSCs after being treated with YODA1 or GsMTx-4 [(B1) the Western blot analysis; (B2) the semiquantitative analysis of LOX, n = 3, one-way ANOVA; (B3) the semiquantitative analysis of LH2, n = 3, one-way ANOVA; (B4) the cell immunofluorescence of LOX; (B5) the semiquantitative analysis of LOX immunofluorescence, n = 6, one-way ANOVA; (B6) the cell immunofluorescence of LH2; (B7) the semiquantitative analysis of LH2 immunofluorescence, n = 6, one-way ANOVA; (B8) the mRNA levels, n = 8, one-way ANOVA]. (C) Protein and mRNA levels of LOX and LH in human SMSCs after being treated with soft or stiff matrix [(C1) the Western blot analysis; (C2) the semiquantitative analysis of LOX, n = 3, unpaired t test; (C3) the semiquantitative analysis of LH2, n = 3, unpaired t test; (C4) the mRNA levels, n = 8, unpaired t test]. (D) Immunofluorescent assessment of LOX expression in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci. (E) Immunofluorescent assessment of LH2 expression in regenerated tissue of PCL scaffold + synovium transplant group and native porcine menisci. *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001; ns, not significant.

Article Snippet: To inhibit phosphorylation of Smad3, human SMSCs were treated with 5 μM SIS3 HCL (S7959, Selleck) for 4 hours.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: NRVMs were transduced with AdV5-CMV-CRYAB R120G -GFP and treated with 3 µM ruxolitinib or DMSO or 100 nM siRNAs targeting either JAK1 (siJak1), JAK2 (siJak2), STAT3 (siStat3) or scramble siRNA (scr). Thereafter, NRVMs were harvested or fixed after 4-6 days. A , Treatment with ruxolitinib or DMSO; Scale bar = 100 µm. B , Transfection with siJak1; Scale bar = 200 µm. C , Transfection with siJak2; Scale bar = 100 µm D , Transfection with siStat3, Scale bar = 20 µm. A , B , C , D , Western blots of protein extracts from treated NRVMs were stained with antibodies directed against indicated proteins. In the representative immunofluorescence images, aggregates are depicted in magenta (CRYAB R120G -GFP), cardiomyocytes in yellow (anti-cardiac troponin I), and nuclei in blue (DAPI). Quantification of aggregates in cardiomyocytes with NIS Elements or ImageJ software. Data were obtained from 1 (Western blot) or 2 (immunofluorescence) NRVM preparations with at least 3 wells per condition and at least 7 images per well for immunofluorescence. Data are depicted as mean ± SEM, and p-values were obtained with the unpaired Student’s t-test. Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transduction, Transfection, Western Blot, Staining, Immunofluorescence, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: A , Heart weight-to-body weight ratio (HW/BW) in 1-, 4- and 7-month-old CRYAB p.Arg120Gly transgenic (R120G) and non-transgenic (NTG) mice. B , Ejection fraction (EF) in 7-month-old R120G and NTG mice. C , Representative Western blots of phosphorylated (P-)STAT3, STAT3 and indicated control in 1-, 4- and 7-month-old R120G and NTG mice. D , P-STAT3/STAT3 quantification of Western blots from 1-, 4- and 7-month-old R120G and NTG mice. E , Stat3 mRNA level determined by RT-qPCR from 7-month-old R120G and NTG mice. Western blot quantification was performed with Image Lab software. Data are depicted as mean ± SEM, and p-values were obtained with the unpaired Student’s t-test. Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transgenic Assay, Western Blot, Control, Quantitative RT-PCR, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: CRYAB p.Arg120Gly transgenic (R120G) and non-transgenic (NTG) mice treated for 3 weeks with 75 mg/kg ruxolitinib (ruxo) or vehicle (veh) twice-daily oral gavage. Transthoracic echocardiography was performed at the start (before, 21-week-old) and the end of the treatment (veh or ruxo, 24-week-old). A , Scheme of experimental outline. B , WB and quantification of phosphorylated (P-) STAT3, STAT3 and indicated controls at the end of treatment. WB quantification was performed with Image Lab software. C, Ejection fraction (EF) and left ventricular mass-to-body weight ratio (LVM/BW) before and after (veh/ruxo) treatment. D , Heart weight-to-body weight ratio (HW/BW) and body weight (BW) at the end of the treatment. E , Representative images and quantification of R120G and NTG mouse heart sections after vehicle or ruxolitinib treatment. CRYAB is depicted in green, cardiomyocytes in purple (anti-cardiac troponin I) and nuclei in blue (DAPI). Scale bar = 10 µm. Quantification of aggregates in cardiomyocytes with NIS Elements software. At least 5 images of 3 mice per group were analyzed. Data are depicted as mean ± SEM, and p-values were obtained with the one- way ( B ) or two-way ANOVA and Tukey’s multiple comparisons post-hoc analysis ( C, D ) or unpaired Student’s t-test ( E ). Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transgenic Assay, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: Heterozygous (het) or homozygous (hom) Jak1 knockout (KO) was induced in MCM-transgenic (TG) mice crossed with CRYAB p.Arg120Gly TG (R120G) or non-transgenic (NTG) mice with tamoxifen chow. Transthoracic echocardiography was performed 26 weeks and hearts were extracted. A , Scheme of experimental outline. B , Jak1 mRNA levels determined by RT-qPCR. C , JAK1 protein levels determined by Western blot and normalized to ACTN2. Western blot quantification was performed with Image Lab software D, Ejection fraction (EF) and left ventricular mass-to-body weight ratio (LVM/BW). E , Heart weight-to-body weight ratio (HW/BW) and body weight (BW). F , Representative images and quantification of mouse heart sections. Aggregates are depicted in green, cardiomyocytes in purple (anti-cardiac troponin I) and nuclei in blue (DAPI). Scale bar = 50 µm. Quantification of aggregates in cardiomyocytes with NIS Elements software. At least 5 images of 3 mice per group were analyzed. Data are depicted as mean ± SEM, and p-values were obtained with the one-way ( B ) or two- way ANOVA ( D , E ) with Tukey’s multiple comparisons post-hoc analysis or unpaired Student’s t-test ( C , F ). Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Knock-Out, Transgenic Assay, Quantitative RT-PCR, Western Blot, Software

Journal: eLife

Article Title: The transcription factor Bach2 negatively regulates murine natural killer cell maturation and function

doi: 10.7554/eLife.77294

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , PureLink RNA Mini Kit , Ambion , Catalog# 12183018A , RNA isolation.

Techniques: Flow Cytometry, Recombinant, Western Blot, Affinity Purification, Cell Isolation, Isolation, Sequencing, Software

Journal: Molecular cell

Article Title: Transcription of nearly all yeast RNA Polymerase II-transcribed genes is dependent on transcription factor TFIID

doi: 10.1016/j.molcel.2017.08.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-V5 Invitrogen Cat # 46-0705 Anti-FLAG M2 Magnetic Beads Sigma-Aldrich Cat # M8823 Rabbit polyclonal anti-Taf1 Hahn lab Rabbit # 3506 Rabbit polyclonal anti-Taf4 Hahn lab Rabbit # 4022 Rabbit polyclonal anti-Taf12 Hahn lab Rabbit # 3434 Rabbit polyclonal anti-Taf3 Hahn lab Rabbit # 4045 Rabbit polyclonal anti-TBP Hahn lab Rabbit # 7370 Rabbit polyclonal anti-H3 Abcam Cat # ab1791 Rabbit polyclonal anti-Taf4 P. Anthony Weil N/A Rabbit polyclonal anti-Taf5 P. Anthony Weil N/A Rabbit polyclonal anti-Taf11 P. Anthony Weil N/A Biological Samples Chemicals, Peptides, and Recombinant Proteins 3-Indoleacetic acid (3-IAA) Sigma-Aldrich I3750; CAS: 87-51-4 Micrococcal Nuclease (MNase) Worthington Biochemical Corporation Cat # NFCB cOmplete Mini, EDTA-free Protease inhibitor cocktail tablets Roche Cat # 11836170001 4-thiouracil Sigma-Aldrich Cat # 440736; CAS: 591-28-6 MTSEA biotin-XX Biotium Cat # 90066 Dynabeads MyOne Streptavidin C1 Invitrogen, ThermoFisher Cat # 65001 AMPureXP beads Beckman Coulter Cat # A63881 EZ-Link HPDP Biotin ThermoFisher Cat # 21341 Rapamycin Euromedex Cat # SYN-1185 Dyanabeads Protein G Thermo Fisher Scientific Cat # 10004D 0.5 mm zirconia disruption beads Research Products International Cat # 9834 Critical Commercial Assays RiboPure yeast kit Ambion, Life Technologies Cat # AM1926 RNeasy MinElute Cleanup Kit Qiagen Cat # 742040 μMACS Streptavidin Kit Miltenyi Biotec Cat# 130-074-101 Deposited Data All sequencing datasets have been uploaded in GEO under accession GSE97081 Western blot original images Mendeley Data file http://dx.doi.org/10.17632/gwtwc4ndpj.1 Experimental Models: Cell Lines Experimental Models: Organisms/Strains S. pombe : Strain background: 972h Laboratory of Gerald Smith ATCC: 24843 S. cerevisiae : Strain background: BY4705 Laboratory of Dan Gottschling ATCC: 200869 Yeast strains, see Table S2 This paper N/A Recombinant DNA Sequence-Based Reagents Primers for RT-qPCR, see Table S1 This paper N/A Software and Algorithms Bowtie2 http://bowtie-bio.sourceforge.net/bowtie2/index.shtml N/A Shell Scripts for Sequence Alignment This paper Perl Scripts for analysis This paper R Scripts for analysis This paper Other Open in a separate window KEY RESOURCES TABLE

Techniques: Magnetic Beads, Recombinant, Protease Inhibitor, Sequencing, Western Blot, Software