Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/elife.12203

Figure Lengend Snippet: Figure 4. Downregulation of CKS1, CyclinA and CDK1 on abrogation of ROCK function. (A) MEFs were treated with H1152 or vehicle for 3 days and the proteome analyzed by quantitative mass spectrometry. Graph shows log ratios of identified proteins. Data are from duplicate experiments of reciprocal SILAC labelling. The ‘Significant-B’ outlier test was used to determine significantly regulated peptides or proteins, using a Benjamini-Hochberg FDR rate of 5%. (B, C) MEFs of different genotypes or treated with indicated inhibitors were immunoblotted for pCDK1, total CDK1, Cyclin A, CKS1, ROCK1, ROCK2 and total ERK as loading control (B). (C) Quantification of western blot analyses. Graphs show protein levels of CDK1, CyclinA and CKS1 divided by total ERK protein levels and SEM in indicated samples. The data are from 5 independent experiments and p-values were calculated using Student’s t-test: * p<0.05, ** p<0.005, *** p<0.001. (D) qPCR analysis of mRNA levels of Cks1b, Cdk1 and Ccna2 in indicated samples. Graphs show average normalized mRNA levels and SD from at least five independent experiments, each carried out in triplicates. p-values were calculated using Student’s t- test: * p<0.05, *** p<0.001. DOI: 10.7554/eLife.12203.013 Figure 4 continued on next page

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phosphop44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKa) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house (Yap et al., 2012b).

Techniques: Mass Spectrometry, Multiplex sample analysis, Control, Western Blot

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/elife.12203

Figure Lengend Snippet: Figure 5. Knockdown of Cdk1 and Cks1b leads to cellular senescence. (A–C) MEFs were infected with either control shRNA or shRNAs targeting Cks1b, Cdk1 and Ccna2. Four days after infection, equal numbers of cells were plated and kept for seven days, allowing colonies to form (A). Five days after infection with shRNAs, cells were also subjected to SA-bGal staining (B) and immunoblotting with antibodies against pCDK1, total CDK1, Cyclin A, CKS1. ROCK2 and total ERK were used as loading control (C). Scale bars are 50 mm. DOI: 10.7554/eLife.12203.016

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phosphop44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKa) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house (Yap et al., 2012b).

Techniques: Knockdown, Infection, Control, shRNA, Staining, Western Blot

Journal: eLife

Article Title: Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

doi: 10.7554/elife.12203

Figure Lengend Snippet: Figure 6. ROCK is essential for tumorigenesis. Cohorts of mice carrying LSL-KrasG12D; LSL-Trp53R270H alleles and the indicated Rock alleles were treated once with Ad-Cre viruses by intranasal inhalation. 24 weeks later, the mice were sacrificed and the lungs were analyzed by histopathology. (A) Graphs show tumor area divided by lung area for indicated genotypes and tumor grades. The data are also displayed as a sum of tumor area divided by lung area for the indicated genotypes. Graphs show average data and SEM. The total number of mice analyzed is indicated in Scatter Plots. (B) Cell lines derived from KrasG12D;Trp53R270H tumors of either Rock1f/f, Rock2f/f, Rock1f/f;Rock2f/f or wild-type genotype were subjected to immunoblotting with indicated antibodies. (C) Isolated lung tumor cell lines of Rock1D/wt;Rock2D/D genotype as well as a cell line which retained both Rock1 alleles (Rock1f/f;Rock2D/D) were treated with Ad-GFP and Ad-Cre-GFP. 3 days after infection, cells were plated and kept for ~5 days allowing colonies to form. (D) Absolute quantification of ROCK1 and 2, using selective reaction monitoring (SRM). Bar graphs show the mean concentration in nM of ROCK1 (black) and ROCK2 (grey) across at least 3 biological replicates from each genetic background. Line 1–3 indicate different cell lines isolated from NSCLCs and Tumors 1–4 represent different micro-dissected tumors. Error bars represent SEM. DOI: 10.7554/eLife.12203.017 The following figure supplement is available for figure 6:

Article Snippet: Secondary antibodies were used at a dilution of 1:10,000 for immunoblotting (LI-COR Biosciences) and 1:500 for immunofluorescence (Alexa Fluor, Life Technologies) The following antibodies were used: p44/42 MAPK (ERK1/2) (1:3000) (Cell Signaling), phosphop44/42 (ERK1/2) (Thr202/Tyr204) (Cell Signaling), GAPDH (1D4) (Novus Biologicals), Rock1 (H-85) (Santa Cruz Biotechnology), Rock2 (ROKa) (BD Biosciences), phospho-MLC (Thr18/Ser19) (Cell Signaling), phospho-MLC (Ser19) for immunofluorescence (Cell Signaling), MRCL3/MRLC2/MYL9 clone (E-4) (Santa Cruz Biotechnology), p21/CIP1 (M-19) (Santa Cruz Biotechnology), Cks1 (Invitrogen), p34cdc2 (Invitrogen), Cdc2 (Y15) (Cell Signaling), CyclinA (CY-A1) (Sigma Aldrich) Inhibitors used were H1152 (Calbiochem/Merck Millipore), GSK429286A (Selleckchem), GSK269962A (Axon Medchem), chroman1 (ApexBio), Blebbistatin + and Blebbistatin +/- (Calbiochem/Merck Millipore), nocodazole (Sigma Aldrich), AT13148 was synthesized in-house (Yap et al., 2012b).

Techniques: Histopathology, Derivative Assay, Western Blot, Isolation, Infection, Quantitative Proteomics, Concentration Assay

Journal: Biological Research

Article Title: Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

doi: 10.1186/s40659-015-0039-2

Figure Lengend Snippet: Efficiency of siRNAs targeting ROCKs and RhoA. a The effectiveness of each siRNA knockdown was evaluated by quantitative polymerase chain reaction analysis. The relative quantities of ROCK1 , ROCK2 , and RhoA mRNAs were compared between each experiment. Top ROCK1 expression was reduced to about 60 and 40 % that of the control using ROCKs-#1 or ROCKs#2 siRNAs. Middle Both combinations of ROCK siRNAs reduced ROCK2 mRNA levels to 40–50 % that of the control. Bottom Each RhoA siRNA reduced RhoA mRNA levels by about 40 % that of the negative control. Data represent the mean ± SEM of 3 independent experiments. NC negative control. Each graph indicates significant difference (* p < 0.01, n = 3). b Immunoblots showing protein expression of ROCK1, ROCK2, RhoA, and β-actin following the incubation of TE-10 cells with siRNA for each target. The expression of each protein was well suppressed, even after 24 h after RNAi treatment. Bars indicate the mean ± SEM of experiments performed in duplicate (RhoA) or triplicate (ROCKs)

Article Snippet: The following primary antibodies were used: rabbit anti-human ROCK1 (1:250; HPA007567; Atlas Antibodies, Stockholm, Sweden), anti-human ROCK2 (1:250; HPA007459; Atlas Antibodies), and anti-human-RhoA (1:100; 26C4; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Expressing, Control, Negative Control, Western Blot, Incubation

Journal: Biological Research

Article Title: Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

doi: 10.1186/s40659-015-0039-2

Figure Lengend Snippet: Migration of TE-10 cell sheets with siRNA knockdown of target mRNAs 72 h after scraping. a Cell migration was quantified over a 48-h period. For each experiment, negative control values were used as the reference. The upper graphs show the migration velocity of the leading edge (Δ LE), while the lower graphs show the velocity of the leading line of the stratified region. Data shown represent the mean ± SEM of at least six independent experiments in which at least three series were stained with Hoechst 33342 and at least 3 series were stained with H2B-GFP. * p < 0.001; ** p = 0.002; *** p = 0.0028; **** p = 0.0044. ANOVA was performed using the original data. NC negative control. b Images of the cell sheets 72 h after scraping under siRNA treatment. First row negative control scrambled siRNA. The shape of the cells in the front row was very similar to that of untreated cells. There was no obvious irregularity in the arrangement of the cells. Second row knockdown of ROCKs by siRNA (ROCK1-#1 and ROCK2-#1). The arrangement of the cells in the leading row was obviously disordered. Many cells in the leading row were small with hypoplastic stress fibers. Several empty spaces between the cells were visible. Third row knockdown of RhoA by siRNA (RhoA-#1). The cells in the simple layer region were smaller than those in the negative control. Many of the cells were small and round, with inconspicuous stress fibers. Green α-tubulin, red β-actin, blue nuclei; scale bar 100 µm. Each inset shows the magnified image of the area surrounded by the interrupted white line on the merged image

Article Snippet: The following primary antibodies were used: rabbit anti-human ROCK1 (1:250; HPA007567; Atlas Antibodies, Stockholm, Sweden), anti-human ROCK2 (1:250; HPA007459; Atlas Antibodies), and anti-human-RhoA (1:100; 26C4; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Migration, Knockdown, Negative Control, Staining

Journal: Biological Research

Article Title: Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

doi: 10.1186/s40659-015-0039-2

Figure Lengend Snippet: Sequences of chimeric siRNAs used in this study

Article Snippet: The following primary antibodies were used: rabbit anti-human ROCK1 (1:250; HPA007567; Atlas Antibodies, Stockholm, Sweden), anti-human ROCK2 (1:250; HPA007459; Atlas Antibodies), and anti-human-RhoA (1:100; 26C4; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Negative Control

Journal: bioRxiv

Article Title: Extracellular Pgk1 enhances neurite outgrowth of motoneurons through Nogo66-independent targeting of NogoA

doi: 10.1101/550921

Figure Lengend Snippet: The molecular pathway mediated by extracellular addition of Pgk1 to enhance neurite outgrowth is independent from the neuronal Nogo66/NgR Pathway. ( A ) Western blot analysis. Protein levels of phosphorylated ROCK2 at Y256 (p-ROCK2-Y256), ROCK2, phosphorylated LimK1 at T508 (p-Limk1-T508), LimK1, phosphorylated Cofilin at S3 (p-Cofilin-S3), total Cofilin, phosphorylated EGFR at Y1173 (p-EGFR-Y1173), EGFR, phosphorylated Akt at S473 (p-Akt-S473) and Akt in neurons cultured with Sol8-vector CM, Sol8-NogoA CM and Sol8-NogoA CM plus Pgk1 addition were all examined, as indicated. The α–tubulin served as internal loading control. ( B-F ) Quantification of protein expression level. The band intensities shown on Western blotting were quantified by ImageJ software. The relative value of each examined protein was used for comparison among the three groups when the value obtained from the Sol8-vector CM group was set as 1. All of the above data were averaged from three independent experiments. Statistical analysis used Student’s t -test (***, p <0.001; **, p <0.01; *, p <0.05).

Article Snippet: The antibodies against NogoA (SC; 1:500), Cofilin (Cell Signaling; CS; 1:1000), phosphorylated Cofilin at S3 (CS; 1:1000), Rho-associated protein kinase 2 (ROCK2) (CS; 1:2000), phosphorylated ROCK2 at Y256 (Rockland, 1:2000), Epidermal growth factor receptor (EGFR) (SC; 1:500), phosphorylated EGFR at Y1173 (CS; 1:2000), Akt (Protein kinase B; PKB) (CS; 1:1000), phosphorylated Akt at S473 (CS; 1:1000), LIM domain kinase 1 (Limk1; SC; 1:500), phosphorylated Limk1 at S323 (Signalway Antibody; SAB; 1:1000), phosphorylated Limk1 at T508 (SAB; 1:500), Phosphoglycerate kinase 1 (Pgk1; Abcam; 1:2000), p21-activated kinase 1 (Pak1) (CS; 1:1000), phosphorylated Pak1 at T423 (CS; 1:1000), P38 mitogen-activated protein kinases (P38) (CS; 1:1000), phosphorylated P38 at T180 (CS; 1:1000), MAP kinase-activated protein kinase 2 (MK2) (CS; 1:1000), phosphorylated MK2 at T334 (CS; 1:1000), Synapsin I (Syn1) (Abcam; 1:1000), Growth Associated Protein 43 (GAP43) (Abcam; 1:1000), Choline acetyltransferase (ChAT) (Abcam; 1:1000), Microtubule-associated protein 2 (MAP2) (Abcam; 1:1000), α-tubulin (SA; 1:5000), Myc (Sigma; 1:2000), Flag (Abcam; 1:5000), mouse-HRP (SC; 1:5000) and rabbit-HRP (SC; 1:5000) were used.

Techniques: Western Blot, Cell Culture, Plasmid Preparation, Expressing, Software

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: Phospho-proteomics, 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, Control, 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