Journal: Journal of Orthopaedic Translation

Article Title: RPL35 downregulated by mechanical overloading promotes chondrocyte senescence and osteoarthritis development via Hedgehog-Gli1 signaling

doi: 10.1016/j.jot.2024.01.003

Figure Lengend Snippet: RPL35 regulates the chondrocyte phenotype in osteoarthritis via the hedgehog (Hh) pathway. (A) Venn diagram analysis of mouse primary chondrocytes treated with 20 % elongation strain loading for 24 h or RPL35 siRNA (Si-RPL35) for 48 h. (B) The bubble plots showing KEGG pathway enrichment data for genes that were up-regulated. (C) Quantitative PCR analysis of RPL35, Gli-1, SMO in chondrocytes treated with 20 % elongation strain loading for 24 h or RPL35 siRNA for 48 h n = 6 per group. (D, E) Immunostaining and quantification of Gli-1, PTCH1 in articular cartilage of mice intra-articularly injected with or without Ad-RPL35 for 4 weeks under mechanical loading. n = 5 per group, Scale bar: 50 μm. (F) Immunofluorescent staining and quantification of SMO in articular cartilage of mice intra-articularly injected with or without Ad-RPL35 for 4 weeks under mechanical loading. n = 5 per group, Scale bar: 50 μm. (G) Western blotting analysis of Gli-1, SMO, PTCH1 in normal primary chondrocytes, RPL35 siRNA-transfected chondrocytes, and mechanical loading chondrocytes treated with or without Ad-RPL35 for 48 h; The GAPDH was used as a loading control. n = 3 per group. (H) Western blotting analysis of Gli-1, MMP13, P16, and P21 in mouse primary chondrocytes treated with RPL35 siRNA for 48 h with or without cyclopamine (10 μM) for 24 h n = 3 per group. *P < 0. 05, **P < 0. 01, ***P < 0. 001 , ns not significant.

Article Snippet: Primary chondrocytes were treated with RPL35 siRNA and 10 μM hedgehog pathway inhibitor cyclopamine (MCE, Monmouth Junction, NJ, USA).

Techniques: Real-time Polymerase Chain Reaction, Immunostaining, Injection, Staining, Western Blot, Transfection, Control

Journal: eBioMedicine

Article Title: Identification of a distal enhancer regulating hedgehog interacting protein gene in human lung epithelial cells

doi: 10.1016/j.ebiom.2024.105026

Figure Lengend Snippet: TGFβ induces HHIP expression in human bronchial epithelial cells. Expression of HHIP in BEAS-2B cells (n = 3 repeats) (a), primary NHBE cells cultured at submerged condition (n = 5 subjects) (b) or in air-liquid interface (ALI) condition (n = 3 subjects) (c), treated with TGFβ1 (5 ng/mL) for indicated durations. (d) Expression of HHIP in primary normal human bronchial epithelial (NHBE) cells treated with TGFβ1 and Hedgehog pathway agonist (n = 5 subjects) or (e) various Hedgehog pathway inhibitors (n = 5 subjects). (f) Expression of HHIP in BEAS-2B cells (n = 3 repeats) and (g) primary NHBE cells (n = 3 subjects with 3 repeats) treated with TGFβ1 (5 ng/mL) and/or TGFβ type I receptor inhibitor SB 431542 (SB, 5 μM). (h) Protein levels of HHIP were measured in BEAS-2B cells treated with TGFβ1 (5 ng/mL) and/or TGFβ type I receptor inhibitor SB 431542 (SB, 5 μM) for 24 and 48 h (Statistical analysis for panel a, b, d, f and g: ordinary one-way ANOVA analysis followed by Dunnett comparisons; panel c: unpaired t -test; panel e: two-way ANOVA followed by Bonferroni comparisons). ∗ P < 0.05, and ∗∗ P < 0.01 comparing to vehicle control group. ## P < 0.01 comparing to TGFβ1-treated group.

Article Snippet: Primary NHBE cells were starved and cultured with basic BEGM medium overnight followed by the treatment with TGFβ1 (R&D Systems, 7754-BH) (5 ng/mL), Hedgehog pathway agonist, SAG (Selleckchem, S6384) (50 nM), an agonist of Smoothened, or various Hedgehog pathway inhibitors, Vismodegib (Selleckchem, S1082) (10 μM), the Smoothened antagonist, Sonidegib (Selleckchem, S2151) (5 μM), and the GLI1 inhibitor, GANT61 (Selleckchem, S8075) (10 μM) for 24 h separately or together as indicated in the figure legends.

Techniques: Expressing, Cell Culture, Control

Journal: Birth defects research

Article Title: High-throughput detection of craniofacial defects in fluorescent zebrafish

doi: 10.1002/bdr2.2127

Figure Lengend Snippet: Hedgehog pathway disruption reduces fli1:EGFP expression at doses that cause craniofacial malformations. (a) Stacked bar graph showing severity scoring of embryos exposed the potent Hedgehog pathway inhibitor BMS-833923. Embryos were exposed from 6–24 hpf and stained at 5 dpf. Number of embryos for each severity are shown (white = apparently normal, light gray = mild, dark gray = moderate, black = severe). (b) Collapsed data showing frequency of malformations for each treatment group. Bar graph of frequency of malformations following ethanol exposure. Statistics = Fischer's exact test for each control-treatment group comparison. (c) Column graph showing fli1:EGFP fluorescence measurement for each embryo (open circles). Black bars show mean fluorescence for each group ± SEM. Sample sizes: control n = 24; 1.56 μM BMS n = 24; 3.125 μM BMS n = 24; 6.25 μM BMS n = 24; and 12.5 μM BMS n = 24. Statistic: two-tailed ANOVA with Tukey's multiple comparison's correction between all groups. * = p < .05, *** = p < .001, **** p < .0001. (d) Images of 5 dpf embryos stained with alcian blue (cartilage) and alizarin red (bone) showing BMS-induced craniofacial defects. RFU = relative fluorescence units. BMS = BMS-833923, Hedgehog pathway inhibitor standard

Article Snippet: The Sonic Hedgehog (Shh) pathway inhibitor standard BMS-833923, CAS# 1059734–66-5, was obtained from Med-Chem Express (Monmouth Junction, NJ).

Techniques: Disruption, Expressing, Staining, Control, Comparison, Fluorescence, Two Tailed Test

Journal: Birth defects research

Article Title: High-throughput detection of craniofacial defects in fluorescent zebrafish

doi: 10.1002/bdr2.2127

Figure Lengend Snippet: Monitoring Hedgehog pathway activity. (a) Column graph showing GliBS:mCherry fluorescence (a marker of Hedgehog pathway activity) for each embryo (open circles) exposed to the indicated concentration of the potent Hedgehog pathway inhibitor standard BMS-833923 from 6–24 hpf. Black bars show mean fluorescence for each group ± SEM. Sample sizes: control n = 21; 1.56 μM BMS n = 24; 3.125 μM BMS n = 24; 6.25 μM BMS n = 16; 12.5 μM BMS n = 11. Many samples, n = 6 at 6.25 μM and n = 13 at 12.5 μM BMS were below fluorescence threshold and were omitted. Statistic: two-tailed ANOVA with Tukey's multiple comparison's correction between all groups. (b) Column graph showing GliBS:mCherry fluorescence for embryos exposed to the environmental teratogen PBO from 6–24 hpf. Black bars show mean fluorescence for each group ± SEM. Sample sizes: control n = 23; PBO 3.125 μM n = 23; PBO 6.25 μM n = 23; PBO 12.5 μM n = 23; PBO 25 μM n = 23; PBO 50 μM n = 16. n = 7 samples exposed to 50 μM PBO were below the fluorescence threshold and were omitted. Statistic: two-tailed ANOVA with Tukey's multiple comparison's correction between all groups. (c) Column graph showing GliBS:mCherry fluorescence for embryos exposed to ethanol from 6–24 hpf. Black bars show mean fluorescence for each group ± SEM. Sample sizes: Statistic: two-tailed ANOVA with Tukey's multiple comparison's correction between all groups. * = p < .05, ** = p < .01, *** = p < .001, **** p < .0001. RFU = relative fluorescence units. BMS = BMS-833923, Hedgehog pathway inhibitor standard. PBO = piperonyl butoxide, environmental Hedgehog pathway inhibitor

Article Snippet: The Sonic Hedgehog (Shh) pathway inhibitor standard BMS-833923, CAS# 1059734–66-5, was obtained from Med-Chem Express (Monmouth Junction, NJ).

Techniques: Activity Assay, Fluorescence, Marker, Concentration Assay, Control, Two Tailed Test, Comparison