Journal: The Journal of Cell Biology

Article Title: Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain

doi: 10.1083/jcb.202007146

Figure Lengend Snippet: Imaging of junctional and apical defects with myo-IIA localization and inhibition by blebbistatin. (A) Deconvolution immunofluorescence staining of myo-IIA shows a similar organization to myo-IIB . Nonmuscle myo-IIA and myo-IIB are localized to aberrant apical contractile fibers. (B) Jeg-3 wild-type and knockout cells were treated with 25 µM blebbistatin for 30 min before immunofluorescence staining of ZO-1 and actin. Images are maximum-intensity projections of apical Z-slices as determined by ZO-1 signal. (C) Quantification of tight junction tortuosity using ZO-1 staining. Each point represents the tortuosity from one tight junction intersection to the next intersection. Center lines represent mean ± SEM; n ≥ 13. Scale bars, 5 µm in A, 10 µm in B.

Article Snippet: Calyculin A (catalog no. BML-El92-0100) was purchased from Enzo Life Sciences, and blebbistatin (catalog no. B592500) was purchased from Toronto Research Chemicals.

Techniques: Imaging, Inhibition, Immunofluorescence, Staining, Knock-Out

Journal: The Journal of Cell Biology

Article Title: Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain

doi: 10.1083/jcb.202007146

Figure Lengend Snippet: pERMs negatively regulate myosin contractility. (A) Representative phase-contrast still images from showing the effect of 10 nM calyculin A on wild-type or knockout cells. (B) Same as A, except for 30-min treatment with 25 µM blebbistatin or transfection to express phosphomimetic ezrin-T567E before calyculin A treatment. Scale bars, 100 µm.

Article Snippet: Calyculin A (catalog no. BML-El92-0100) was purchased from Enzo Life Sciences, and blebbistatin (catalog no. B592500) was purchased from Toronto Research Chemicals.

Techniques: Knock-Out, Transfection

Journal: Nature Cell Biology

Article Title: Ex vivo imaging reveals the spatiotemporal control of ovulation

doi: 10.1038/s41556-024-01524-6

Figure Lengend Snippet: a , The relative volume of DMSO- and mifepristone-treated follicles. Total n = 26; individual groups: DMSO n = 13, mifepristone n = 13. b , The relative volume of DMSO- and JKC-301-treated follicles. Total n = 21; individual groups: DMSO n = 10, JKC-301 n = 11. c , The relative volume at 11 h of DMSO-, mifepristone- and JKC-301-treated follicles. P values: control versus mifepristone = 6.13 × 10 −6 , control versus JKC-301 = 1.29 × 10 −10 . d , The ovulation rate in DMSO-, mifepristone- and JKC-301-treated follicles. P values: control versus mifepristone = 0.000027, control versus JKC-301 = 0.008636. In c , d , total n = 47; individual groups: DMSO n = 23, mifepristone n = 13, JKC-301 n = 11, as shown above the graphs. e , Upregulated GO terms in smooth muscle cells from 6 to 9 h. FDR, false discovery rate. f , A condensed heatmap of mitochondrial respiratory chain gene expression in smooth muscle cells during follicle contraction. g , The relative volume of DMSO- and FCCP-treated follicles. DMSO n = 15, FCCP n = 15. OXPHOS, oxidative phosphorylation. h , The change in relative volume between 9 h and 11 h of DMSO- and FCCP-treated follicles. P = 6.56 × 10 −6 . i , The ovulation rate in DMSO- and FCCP-treated follicles. P = 0.00001. In h , i , total n = 30; individual groups: DMSO n = 15, FCCP n = 15, as shown above the graphs. j , Upregulated GO terms in smooth muscle cells from 9 to 12 h. FDR, false discovery rate. k , A heatmap of actomyosin contraction gene expression in smooth muscle cells during follicle contraction. l , The relative volume of control ((+) blebbistatin inactive enantiomer) and blebbistatin ((−) blebbistatin active enantiomer)-treated follicles. Total n = 24; individual groups: Control n = 13, blebbistatin n = 11. m , The relative volume of DMSO- and Y-27632-treated follicles. Total n = 28; individual groups: DMSO n = 14, Y-27632 n = 14. n , The relative volume at 11 h of control and blebbistatin- and Y-27632-treated follicles. P values: control versus blebbistatin = 1.98 × 10 −14 , control versus Y-27632 = 7.94 × 10 −21 . Total n = 66; individual groups: Control n = 41, blebbistatin n = 11, Y-27632 n = 14, as shown above the graph. o , The ovulation rate in control and blebbistatin- and Y-27632-treated follicles. P values: control versus blebbistatin = 0, control versus Y-27632 = 0.000016. Total n = 69; individual groups: DMSO n = 41, blebbistatin n = 13, Y-27632 n = 15, as shown above the graph. p , Representative images of α-SMA and phospho-Myl9 staining in cryosectioned follicles collected at 12 h ± Y-27632. Scale bar, 100 μm. q , Quantification of phospho-Myl9 mean fluorescence intensity in the outer follicle normalized to the mean fluorescence intensity of α-SMA at 6, 9 and 12 h ± Y-27632. 6 h DMSO n = 88, 9 h DMSO n = 109, 9 h Y-27632 n = 53, 12 h DMSO n = 55, 12 h Y-27632 n = 50, as shown above the graph. P values: 9 h control versus Y-27632 = 0.03368, 12 h control versus Y-27632 = 2.37 × 10 −8 . Data in a – d , g – i and l – q are representative of two independent biological repeats. Data in e , f , j and k are representative of three independent biological repeats. In a , b , g , l and m , the thick lines show the mean, and the lighter shadows show the s.e.m. In c , h , n and q : **** P < 0.0001, *** P < 0. 001, ** P < 0.01, n.s. non-significant, two-sided unpaired t -test; box, 25–75%; whiskers, 5–95%; centre, median; centre box, mean. In d , i and o : chi-square test (two-sided) with Yates’s correction. All times are relative to hCG addition.

Article Snippet: Drugs used in this study include 4-MU (0.5 and 1 mM; Sigma-Aldrich, M1381), mifepristone (100 μM; Sigma-Aldrich, M8046), JKC-301 (10 μM; Sigma-Aldrich, SCP0141), FCCP (10 μM; Sigma-Aldrich, C2920), blebbistatin (−) and (+) (100 μM; Tocris, 1852 and 1853), Y-27632 (30 μM; Tocris, 1254) and SB-3CT (250 μM; Sigma-Aldrich, S1326).

Techniques: Control, Gene Expression, Phospho-proteomics, Staining, Fluorescence

Journal: Nature Cell Biology

Article Title: Ex vivo imaging reveals the spatiotemporal control of ovulation

doi: 10.1038/s41556-024-01524-6

Figure Lengend Snippet: ( a ) Graph showing absolute volume, non-normalised, in DMSO and mifepristone-treated follicles. ( b ) Graph showing volume normalised at 6.5 h post hCG addition in DMSO and mifepristone-treated follicles. Panels (a,b): total n = 26; individual groups: DMSO n = 13, mifepristone n = 13. ( c ) Graph showing absolute volume, non-normalised, in DMSO and JKC-301-treated follicles. ( d ) Graph showing volume normalised at 6.5 h post hCG addition in DMSO and JKC-301-treated follicles. Panels (c,d): total n = 21; individual groups: DMSO n = 10, JKC-301 n = 11. ( e ) Graph showing absolute volume, non-normalised, in DMSO and FCCP-treated follicles. ( f ) Graph showing volume normalised at 6.5 h post hCG addition in DMSO and FCCP-treated follicles. Panels (e,f): total n = 30; individual groups: DMSO n = 15, FCCP n = 15. ( g ) Graph showing absolute volume, non-normalised, in control and blebbistatin-treated follicles. ( h ) Graph showing volume normalised at 6.5 h post hCG addition in control and blebbistatin-treated follicles. Panels (g,h): total n = 24; individual groups: DMSO n = 13, blebbistatin n = 11. ( i ) Graph showing absolute volume, non-normalised, in DMSO and Y-27632-treated follicles. ( j ) Graph showing volume normalised at 6.5 h post hCG addition in DMSO and Y-27632-treated follicles. Panels (i,j): total n = 28; individual groups: DMSO n = 14, Y-27632 n = 14. ( k ) Representative whole follicle image of α-SMA and phospho-Myl9 staining of cryosectioned follicles collected at 6, 9, and 12 h post hCG addition. Here, the 9 hour timepoint is shown. ( l ) Representative image of α-SMA and phospho-Myl9 staining of cryosectioned whole ovaries collected at 6, 9, and 12 h post hCG addition. Here, the 12 h timepoint is shown. Data in panels (a,b,c,d,e,f,g,h,i,j,k,l) representative of 2 independent biological repeats. In panels (a,b,c,d,e,f,g,h,i,j): thin traces show raw values from individual follicles; thick traces show the mean.

Article Snippet: Drugs used in this study include 4-MU (0.5 and 1 mM; Sigma-Aldrich, M1381), mifepristone (100 μM; Sigma-Aldrich, M8046), JKC-301 (10 μM; Sigma-Aldrich, SCP0141), FCCP (10 μM; Sigma-Aldrich, C2920), blebbistatin (−) and (+) (100 μM; Tocris, 1852 and 1853), Y-27632 (30 μM; Tocris, 1254) and SB-3CT (250 μM; Sigma-Aldrich, S1326).

Techniques: Control, Staining

Journal: Biochemical and biophysical research communications

Article Title: The Molecular Mechanism of Induction of Unfolded Protein Response by Chlamydia

doi: 10.1016/j.bbrc.2018.11.034

Figure Lengend Snippet: Figure 3a: Total proteins were prepared from Ct. L2 infected and non-infected HeLa cells 48hr p.i. were analyzed by western blotting and probed with an anti-IRE1α[p-Ser724] antibody. Lane 1: non-infected cells; Lane 2: Ct. L2-infected cells; Lane 3: Ct. L2-infected cells treated with 20µM of ML7-hydrochloride (ML7); Lane 4: Ct. L2-infected cells treated with 50µM of (s)-(-)-Blebbistatin (Bleb). Figure 3b: (A 1−3) Microscope image of fluorescent-stained Ct. L2 inclusions (green dots) in HeLa cell monolayer treated with: DMSO as a control for the effect of solvent used in reconstituting inhibitors (A1); 20µM of ML7-hydrochloride (A2); 50µM of (s)-(-)-Blebbistatin (A3). (B) A bar graph presentation of the number of Chlamydia inclusions counted in the three different conditions of DMSO control (), 20µM ML7-hydrochloride () and 50µM (s)-(-)-Blebbistatin (). Treatment with 20µM ML7-hydrochloride shows a highly statistical significant reduction in number and size of inclusions with a two-tailed P-value 0.0001. Treatment with 50µM (s)-(-)-Blebbistatin shows a highly statistical significant reduction in number of inclusions with a two-tailed P-value 0.003. Results are from three independent experiments.

Article Snippet: In vitro studies for the effect of inhibition of myosin light chain kinase (MLCK) and Myosin-II ATPase activities on UPR pathways activation and Chlamydia replication were performed by treating cultures with 20µM of ML-7 hydrochloride or 50µM of (S)-(-)-Blebbistatin (Santa Cruz Biotec., Inc, Dallas, TX, USA) or equal amount of DMSO solution that was used in preparing each inhibitor in the infection medium.

Techniques: Infection, Western Blot, Microscopy, Staining, Two Tailed Test