Journal: Nature Communications

Article Title: SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways

doi: 10.1038/s41467-023-39600-4

Figure Lengend Snippet: a Quantitative phosphoproteomic analysis in 16HBE cells treated with IL-13 (100 ng/ml) at 6 h post and during pretreatment with 2-D08 (30 μM). The bar graph shows the quantitative results of phosphorylated sites. b Enrichment and analysis of the protein domains of differentially expressed phosphorylated peptides. c The altered phosphorylation levels on the key phosphorylation sites of ROCK2 upon IL-13 or/and 2-D08 treatment. d , e Western analyses in 16HBE cells treated with IL-13 at 6 h post and during treatment with 2-D08 ( n = 3, P values: 0.0002, 0.0015, 0.0016; <0.0001, 0.0076, 0.0003). f – m Lungs and bronchi from Fig. and Fig. were subjected to immunofluorescence staining and western analyses, respectively, each n = 6 or 3, P values: <0.0001, <0.0001, <0.0001 ( g ); 0.0015, 0.009, 0.0023 ( i ); <0.0001, <0.0001, <0.0001 ( k ); 0.0022, 0.0364, 0.0089 ( m ). Scale bar, 5 μm. Mean ± SD, One-way ANOVA and Tukey-Kramer multiple comparisons test, + P < 0.05, ++, ** P < 0.01. Source data are provided as a Source Data file.

Article Snippet: The conditional caRhoA ( caRhoA +/− ) knock-in mouse strain with genetic background of C57BL/6J was generated by Cyagen Biosciences (Santa Clara, CA) as described previously , and the conditional Rock2 K1007R/+ knock-in founders with genetic background of C57BL/6 J were generated by CRISPR/Cas9 at Cyagen Biosciences as described previously .

Techniques: Western Blot, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways

doi: 10.1038/s41467-023-39600-4

Figure Lengend Snippet: a CC10-Cre and CC10-Cre; caRhoA +/- mice at 8 weeks of age were peritoneally injected with tamoxifen at 200 mg/kg on day 0, 1, 2, 3, and 4, and then intratracheally received 2-D08 at 10 or 30 mg/kg on day 9, 11, 13, and 15. Mice were euthanized on day 16 for the following analyses. b Cell counting and classification in BALFs. c – f H&E and PAS staining (scale bar, 10 μm), and immunostaining for Muc5AC and p-ROCK2 (scale bar, 5μm) in lung sections and their semi-quantification. P values: <0.0001, 0.0107, <0.0001 ( d ); <0.0001, 0.0134, <0.0001 ( e ); <0.0001, 0.0135, <0.0001 ( f ). g , h Western analyses and semi-quantification for bronchi ( P values: 0.0008, 0.0019, 0.0004). Mean ± SD, n = 6, One-way ANOVA and Tukey-Kramer multiple comparisons test, * , + P < 0.05, ** , ++ P < 0.01. Source data are provided as a Source Data file.

Article Snippet: The conditional caRhoA ( caRhoA +/− ) knock-in mouse strain with genetic background of C57BL/6J was generated by Cyagen Biosciences (Santa Clara, CA) as described previously , and the conditional Rock2 K1007R/+ knock-in founders with genetic background of C57BL/6 J were generated by CRISPR/Cas9 at Cyagen Biosciences as described previously .

Techniques: Injection, Cell Counting, Staining, Immunostaining, Western Blot

Journal: Nature Communications

Article Title: SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways

doi: 10.1038/s41467-023-39600-4

Figure Lengend Snippet: a , b Western analyses in 293T cells transfected with Myc-SUMO1 for 24 h or the indicated times. c Co-immunoprecipitation experiments using a control IgG or a ROCK2 antibody in 16HBE cells and mouse primary bronchial epithelial cells (MPBEs). d Co-immunoprecipitation experiments using a Flag antibody in 16HBE cells transfected with Flag-ROCK2 in combination with Myc-SUMO1 in the presence of scramble or UBC9 shRNA. e In vitro SUMOylation assays in a reaction mixture containing ROCK2 recombinant protein, E1, E2, and SUMO1 and incubated at 37 °C or 4 °C for 60 min, followed by western analyses. f Western analyses in 293T cells 24 h after transfection with vector, wild-type (WT) ROCK2 or ROCK2 variants (K → R). g Co-immunoprecipitation experiments using a Flag antibody in 16HBE cells transfected with Myc-SUMO1 and Flag-ROCK2/Flag-ROCK2(K1007R). h Western analyses in 293T cells at 24 h post-transfection with or without Myc-SUMO1, UBC9 shRNA, and Flag-ROCK2 /Flag-ROCK2(K1007R). i In vitro SUMOylation assays in a reaction mixture containing ROCK2(WT) or ROCK2(K1007R) recombinant protein, E1, E2, and SUMO1, followed by western analyses. j , k Western analyses in 293 T cells transfected with Flag-ROCK2(WT or K1007R) in the presence of IL-13 stimulation for 6 h or of Myc-caRhoA. l Co-immunoprecipitation experiments using a Flag antibody in 16HBE cells transfected with HA-RhoA and Flag-ROCK2(WT or K1007R) after IL-13 treatment for 6 h. m 16HBE cells transfected with Flag-ROCK2(WT or K1007R), were subjected to GST pull-down assays with Rhotekin-RBD-coated beads, followed by western analyses. Experiments were repeated independently at least three times with similar results. Source data are provided as a Source Data file.

Article Snippet: The conditional caRhoA ( caRhoA +/− ) knock-in mouse strain with genetic background of C57BL/6J was generated by Cyagen Biosciences (Santa Clara, CA) as described previously , and the conditional Rock2 K1007R/+ knock-in founders with genetic background of C57BL/6 J were generated by CRISPR/Cas9 at Cyagen Biosciences as described previously .

Techniques: Western Blot, Transfection, Immunoprecipitation, shRNA, In Vitro, Recombinant, Incubation, Plasmid Preparation

Journal: Nature Communications

Article Title: SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways

doi: 10.1038/s41467-023-39600-4

Figure Lengend Snippet: a , b Western analyses in 293T cells at 48 h post transfection with Myc-PIAS1, 2, 3, 4 or siRNAs of PIAS1, 2, 3, 4 in combination with or without Myc-SUMO1. c Western analyses in 293T cells at 48 h post transfection with or without Myc-PIAS1 and PIAS1 siRNA. d Co-immunoprecipitation experiments in 293T cells at 48 h post transfection with or without Myc-PIAS1 and Flag-ROCK2. e , f Western and co-immunoprecipitation analyses in 293T cells at 48 h post transfection with or without Myc-PIAS1 and Flag-ROCK2(WT or K1007R). g IHC of PIAS1 in human healthy bronchial sections ( n = 4, left: experimental group, right: negative control group, scale bar, 2 μm). h , i Immunostaining of CC10, PIAS1, DAPI and semi-quantification in BALF cells from children with FBA or asthma ( P value: <0.0001, scale bar, 10 μm). j – m Lungs or bronchi from NS- and OVA-challenged mice were subjected to IHC ( j ), qPCR ( k , P value: 0.0127), and western analyses ( l ) and their semi-quantification ( m , P value: 0.0093). Scale bar, 10 μm. n – q Mice were intratracheally instilled with lentiviral scramble- or PIAS1-shRNA and then with IL-13. Lungs and bronchi were subjected to H&E (scale bar, 10 μm), PAS (scale bar, 10 μm), Muc5AC (scale bar, 10 μm) and p-ROCK2 (scale bar, 5 μm) staining ( n , o , P values: <0.0001, <0.0001; <0.0001, <0.0001; <0.0001, 0.0014) and western analyses ( p , q , P values: <0.0001, 0.0002; 0.0002, 0.0031). Mean ± SD, n = 4, unpaired two-tailed Student’s t test or One-way ANOVA and Tukey-Kramer multiple comparisons test, * , + P < 0.05, ** , ++ P < 0.01. Experiments were repeated independently at least three times with similar results. Source data are provided as a Source Data file.

Article Snippet: The conditional caRhoA ( caRhoA +/− ) knock-in mouse strain with genetic background of C57BL/6J was generated by Cyagen Biosciences (Santa Clara, CA) as described previously , and the conditional Rock2 K1007R/+ knock-in founders with genetic background of C57BL/6 J were generated by CRISPR/Cas9 at Cyagen Biosciences as described previously .

Techniques: Western Blot, Transfection, Immunoprecipitation, Negative Control, Immunostaining, shRNA, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways

doi: 10.1038/s41467-023-39600-4

Figure Lengend Snippet: a , b Rock2 K1007R/K1007R , CC10-Cre; Rock2 K1007R/+ and CC10-Cre; Rock2 K1007R/K1007R mice were sensitized and challenged with OVA as described in Fig. , and bronchi were then subjected to western analyses and semi-quantification (each n = 3, P values: 0.0016, 0.0486, 0.0073). c , d Lungs (each n = 6) were subjected to paraffin-embedded sectioning, H&E and PAS staining, immunostaining for Muc5AC and p-ROCK2 ( c , scale bar, 10 μm), and their semi-quantification ( d , P values: <0.0001, 0.0001, <0.0001; <0.0001, 0.0115, <0.0001; <0.0001, <0.0001, <0.0001). e , f BALF cell counting and classification ( e , P values: <0.0001, <0.0001, <0.0001, <0.0001, <0.0001; 0.0006, 0.0437, 0.0159; <0.0001, <0.0001, 0.0006, 0.0155) and methacholine-provoked airway hyperreactivity ( f , each n = 6, P values: 0.0002, 0.0001, <0.0001, <0.0001, <0.0001; 0.049, 0.0399, 0.0073, 0.0095; 0.0196, 0.0019, 0.0035, 0.0002, 0.0003). g – l Rock2 K1007R/K1007R , CC10-Cre; Rock2 K1007R/+ and CC10-Cre; Rock2 K1007R/K1007R mice were intratracheally instilled with or without IL-13 as described in Fig. , and lungs or bronchi were subjected to H&E and PAS staining and immunostaining for Muc5AC and p-ROCK2 ( g – j , each n = 6, P values: <0.0001, 0.0001, <0.0001; <0.0001, 0.0036, <0.0001; <0.0001, <0.0001, <0.0001) or western analyses ( k , l , each n = 3, P values: 0.0015, 0.0236, 0.0034). Scale bar, 10 μm. Mean ± SD, One-way ANOVA and Tukey-Kramer multiple comparisons test, * , + P < 0.05, ** , ++ P < 0.01. Source data are provided as a Source Data file.

Article Snippet: The conditional caRhoA ( caRhoA +/− ) knock-in mouse strain with genetic background of C57BL/6J was generated by Cyagen Biosciences (Santa Clara, CA) as described previously , and the conditional Rock2 K1007R/+ knock-in founders with genetic background of C57BL/6 J were generated by CRISPR/Cas9 at Cyagen Biosciences as described previously .

Techniques: Western Blot, Staining, Immunostaining, Cell Counting

Journal: The Journal of Neuroscience

Article Title: Role of DNA Methylation in the Nucleus Accumbens in Incubation of Cocaine Craving

doi: 10.1523/JNEUROSCI.3053-14.2015

Figure Lengend Snippet: Cue-induced cocaine- or sucrose-seeking after intra-NAc injection of the ESR1 agonist PPT or the CDK5 inhibitor roscovitine. Tracks represent significant methylation probe fold differences (Log 2) between RG108-treated and aCSF-injected animals, as determined by MeDIP analysis for the esr1 (A) and cdk5 (B) genes (the 3 and 5 first exons are shown in red for esr1 and cdk5, respectively). esr1 and cdk5 mRNA levels after RG108 treatment are displayed (results identical to Fig. 6A). A, B, Rats were trained for cocaine self-administration, and on days 29 and 30 d withdrawal received intra-NAc injections of PPT (an ESR1 agonist) or roscovitine (a CDK5 inhibitor). During the extinction test on day 30, PPT-treated (A) and roscovitine-treated (B) rats showed a decrease in the mean number of active lever presses (±SEM). ****p < 0.0001 (Student's t test; PPT, n = 4 per group). **p = 0.0027 (Student's t test; roscovitine, n = 6 per group). Both treatments significantly affected the number of active lever presses, whereas no change in active lever presses was observed in the corresponding control groups. The number of inactive lever presses for treatment and control groups was <7% of active lever presses of controls, and not significantly different between groups (3.7 ± 1.5 for roscovitine vs 6.6 ± 1.5 for aCSF, 2.5 ± 0.6 for PPT vs 4.8 ± 1.4 for aCSF; p > 0.05, Student's t test). No difference in locomotion was found for PPT-treated (A) or roscovitine-treated rats (B), compared with aCSF-treated rats. C, Rats were trained for self-administration of sucrose (10%; 0.13 ml/infusion, 3 h/d, 10 d), and on day 21 of withdrawal received intra-NAc PPT or roscovitine and were then subjected to an extinction test. PPT-treated and roscovitine-treated rats showed a decrease in the mean number of active lever presses (±SEM) compared with aCSF-treated rats. ***p < 0.001 (one-way ANOVA followed by Student-Newman-Keuls' test). The number of inactive lever presses for treatment and control groups was <10% of active lever presses of controls, and not significantly different between groups (4.9 ± 0.8 for roscovitine, 5 ± 1.1 for PPT, 10 ± 3.9 for control; p > 0.05, Student's t test).

Article Snippet: Antibodies used were anti-H2B (1:1000; Abcam; Ab1790), anti-NMDAR2A (1:1000; Millipore; AB1555P), anti-ESR1 (1:1000; Millipore; 04820), anti-GluR1 (GluA1) (1:500; Millipore; ABN241), and HRP-conjugated anti-rabbit (1:80,000; Sigma-Aldrich; A0545).

Techniques: Injection, Methylation, Methylated DNA Immunoprecipitation