Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that ROCK1 mean intensities within circumferential rings (CRs) are similar between WT and LUZP1 KO cells (40.87 ± 9.95 arbitrary units [a.u.] [WT] vs. 39.48 ± 6.04 a.u. [LUZP1 KO]). n = 3. P = 0.54 (Mann–Whitney U test). Bars and error bars represent the mean ± standard deviation (SD). In vitro myosin light chain (MLC) phosphorylation assay using 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the ppMLC/MLC ratio relative to the control showed that LUZP1 did not change the ratio (1.00 [1 st lane, control] vs. 1.13 ± 0.24 [2 nd lane] vs. 1.01 ± 0.44 [3 rd lane] vs. 1.08 ± 0.73 [4 th lane]). n = 4. P = 0.49 (Kruskal–Wallis test). Bars and error bars represent the mean ± SD. IB, immunoblotting. Representative confocal micrographs of co‐cultures of Venus‐LUZP1‐expressing LUZP1 KO (REV) and LUZP1 KO Eph4 cells treated with 100 nM calyculin A for 30 min. Scale bar, 10 μm. Bar plots with dot density plots showing that calyculin A reversed the difference in ppMLC levels within CRs between REV and LUZP1 KO cells (control, 21.14 ± 16.80 a.u. [WT] vs. 3.10 ± 1.72 a.u. [LUZP1 KO]; calyculin A, 25.24 ± 10.54 a.u. [WT] vs. 20.65 ± 5.62 a.u. [LUZP1 KO]; washout, 22.09 ± 7.90 a.u. [WT] vs. 7.92 ± 4.01 a.u. [LUZP1 KO]). ** P < 0.01 (Mann–Whitney U test). Bars and error bars represent the mean ± SD. n = 3. Representative immunoblot of WT, LUZP1 KO, and Venus‐LUZP1‐expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A (WT, 1.00 [control] vs. 1.40 ± 0.06 [calyculin A] vs. 1.14 ± 0.33 [washout]; KO, 0.09 ± 0.04 [control] vs. 1.49 ± 0.06 [calyculin A] vs. 0.81 ± 0.99 [washout]; REV, 2.06 ± 1.78 [control] vs. 1.82 ± 1.50 [calyculin A] vs. 1.80 ± 1.14 [washout]). n = 3. Bars and error bars represent the mean ± SD. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Knock-Out, MANN-WHITNEY, Standard Deviation, In Vitro, Phosphorylation Assay, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: A schematic drawing of myosin phosphatase. Myosin phosphatase consists of PP1c β/δ, myosin phosphatase targeting subunit 1 (MYPT1), and a small 20‐kDa regulatory subunit (M20). PP1c β/δ represents a catalytic subunit responsible for dephosphorylating myosin light chain (MLC), whereas MYPT1 targets myosin phosphatase to MLC by binding both PP1c β/δ and MLC. Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that PP1c mean intensities within CRs are similar between WT and LUZP1 KO cells (28.68 ± 9.60 arbitrary units [a.u.] [WT] vs. 25.04 ± 9.47 a.u. [LUZP1 KO]). P = 0.09 [Mann–Whitney U test]. n = 3. Bars and error bars represent the mean ± standard deviation (SD). Co‐immunoprecipitation of HA‐PP1c β/δ and GFP‐LUZP1. LUZP1 binds to PP1c β/δ. IB, immunoblotting. In vitro MLC phosphorylation assay using 1 μg GST‐PP1c β/δ in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the di‐phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 upregulated ppMLC/MLC levels in a dose‐dependent manner (1.00 [1 st lane, control] vs. 1.27 ± 0.33 [2 nd lane] vs. 1.76 ± 0.68 [3 rd lane] vs. 2.53 ± 1.65 [4 th lane] vs. 2.93 ± 2.45 [5 th lane]). n = 3 or 6. ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. In vitro Merlin phosphorylation assay using 1 μg GST‐PP1c β/δ, 100 ng GST‐Merlin, 2 pg p21‐activated kinase 1 (PAK1), and 5 μg GST‐LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 upregulated pMerlin/Merlin levels (0.23 ± 0.15 [1 st lane] vs. 1.00 [2 nd lane, control] vs. 0.32 ± 0.17 [3 rd lane] vs. 0.97 ± 0.42 [4 th lane] vs. 1.25 ± 0.39 [5 th lane]). n = 4 or 9. * P < 0.05, ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 3 rd lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among ppMLC, LUZP1, and myosin phosphatase at tight junction (TJ)‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Binding Assay, Knock-Out, MANN-WHITNEY, Standard Deviation, Immunoprecipitation, Western Blot, In Vitro, Phosphorylation Assay

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Box plots with dot density plots showing the ratio of the apical area/basal area in co‐cultures of Venus‐LUZP1‐expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells (REV, 0.65 ± 0.16 [control] vs. 0.90 ± 0.18 [nocodazole] vs. 0.64 ± 0.16 [washout]; KO, 1.30 ± 0.17 [control] vs. 1.07 ± 0.13 [nocodazole] vs. 1.32 ± 0.19 [washout]). ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). The solid lines represent the medians, and the boxes represent the interquartile ranges. The error bars extending from the box represent the data within 1.5 times of the interquartile range. Representative confocal micrographs of co‐cultures of LUZP1‐expressing wild‐type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di‐phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm. Bar plots with dot density plots showing that ppMLC levels within CRs were significantly downregulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment (WT, 21.43 ± 6.96 arbitrary units [a.u.] [control] vs. 17.67 ± 5.40 a.u. [nocodazole] vs. 20.84 ± 7.19 a.u. [washout]; KO, 8.74 ± 1.71 a.u. [control] vs. 8.67 ± 1.89 a.u. [nocodazole] vs. 7.96 ± 2.35 a.u. [washout]). n = 3. ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). Bars and error bars represent the mean ± standard deviation (SD). In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, 1 μg GST‐protein phosphatase 1c β/δ (PP1c β/δ), and 0–5 μg GST‐LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1‐mediated inhibition of PP1c β/δ (1.00 [1 st ‐lane, control] vs. 1.42 ± 0.59 [2 nd ‐lane] vs. 1.72 ± 0.76 [3 rd ‐lane] vs. 1.99 ± 0.56 [4 th ‐lane] vs. 1.14 ± 0.37 [5 th ‐lane] vs. 2.87 ± 1.51 [6 th ‐lane] vs. 2.74 ± 1.19 [7 th ‐lane] vs. 2.50 ± 0.88 [8 th ‐lane]). n = 6. * P < 0.05 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among MTs, ppMLC, LUZP1, and myosin phosphatase at TJ‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Expressing, Knock-Out, Standard Deviation, In Vitro, Phosphorylation Assay, Inhibition

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet:

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Transfection, Protease Inhibitor, Purification, Western Blot, Blocking Assay, Software, Imaging, Modification

Journal: Journal of Neuroinflammation

Article Title: Orexin A alleviates neuroinflammation via OXR2/CaMKKβ/AMPK signaling pathway after ICH in mice

doi: 10.1186/s12974-020-01841-1

Figure Lengend Snippet: Western blot analysis showing anti-inflammatory effects of OXA. a Representative protein bands and the quantitative analysis of b ratio of p-CaMKKβ/CaMKKβ and c ratio of p-AMPK/AMPK at 24 h after ICH. The expression of p-CaMKKβ and p-AMPK was upregulated after ICH and was further elevated by OXA. This effect was reversed with STO-609 (p-CaMKKβ inhibitor). Consequently, the inflammatory factors including d p-NFκB, e IL-1β, and f TNFα were increased after ICH and were significantly suppressed by OXA. Consistently, this effect was reversed with STO-609. One-way ANOVA, * p < 0.05 vs. sham group, # p < 0.05 vs. vehicle group, @ p < 0.05 vs. OXA group. Error bars represent mean ± SD, n = 6 per group

Article Snippet: CaMKKβ inhibitor, STO-609 (S8274, Selleck Chem, Houston, TX, USA), was dissolved in 5% dimethyl sulfoxide (DMSO) to yield a drug solution with a concentration of 5 mg/ml, and 100 μl of the drug solution was injected intraperitoneally ( i.p. ) 1 h after ICH induction [ ].

Techniques: Western Blot, Expressing

Journal: Journal of Neuroinflammation

Article Title: Orexin A alleviates neuroinflammation via OXR2/CaMKKβ/AMPK signaling pathway after ICH in mice

doi: 10.1186/s12974-020-01841-1

Figure Lengend Snippet: ELISA tests to evaluate inflammation-related cytokines with OXA treatment. The levels of pro-inflammatory cytokines including a IL-1β, b TNFα, c IL-6, and d IL-12 were significantly downregulated with OXA treatment, and the levels of anti-inflammatory cytokines including e IL-4 and f IL-10 were significantly upregulated with OXA treatment evaluated at 24 h after ICH. The effects of OXA were reversed by CaMKKβ inhibitor (STO-609) and OXR2 inhibitor (JNJ-10397049) but not OXR1 inhibitor (SB-334867). One-way ANOVA, * p < 0.05 vs. sham group, # p < 0.05 vs. vehicle group, @ p < 0.05 vs. OXA group. Error bars represent mean ± SD, n = 6 per group

Article Snippet: CaMKKβ inhibitor, STO-609 (S8274, Selleck Chem, Houston, TX, USA), was dissolved in 5% dimethyl sulfoxide (DMSO) to yield a drug solution with a concentration of 5 mg/ml, and 100 μl of the drug solution was injected intraperitoneally ( i.p. ) 1 h after ICH induction [ ].

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy

doi: 10.1371/journal.pone.0130477

Figure Lengend Snippet: To investigate the role of the cross talk between CaMKII and ERK on cardiac hypertrophy, we assessed the effect of CaMKII inhibition on PE-induced ERK activation. H9C2s were pretreated with KN93 (5 μmol/L) or AntCaNtide (10 μmol/L) and tat-CN17β (10 μmol/L) for 30 min and then stimulated with PE (100 nmol/L for 15 min.). A: H9C2 total cell lysates were analyzed by Western blotting (WB) for phosphothreonine 286 CaMKII (pCaMKII) and total CaMKII (CaMKII), with specific antibodies. Data from immunoblots were quantified by densitometric analysis (DA). pCaMKII levels were corrected by total CaMKII densitometry. * = p<0.05 vs Ctrl; # = p<0.05 vs PE. B: Total H9C2 cell extracts were be subjected to WB analysis to visualize phosphotyrosine (pERK1/2) and total ERK (ERK1/2) cell content using anti-pERK1/2 or anti-total ERK1/2 antibodies. pERK1/2 levels were corrected by total ERK1/2 densitometry. * = p<0.05 vs Ctrl; # = p<0.05 vs PE. C: H9C2 total lysates were immunoprecipitated using anti-CaMKII antibody. The protein samples underwent WB procedure to visualize ERK and evaluate the association with CaMKII. The experiments were normalized by WB for total CaMKII. ERK1/2 levels were corrected by CaMKII densitometry. * = p<0.05 vs Ctrl; # = p<0.05 vs PE. D: To confirm the interaction between CaMKII and ERK in H9C2, total cell lysates were immunoprecipitated using anti-ERK1/2 antibody, and subjected to WB using anti-CAMKII antibody. CaMKII levels were corrected by ERK1/2 densitometry. * = p<0.05 vs Ctrl; # = p<0.05 vs PE. E: Nuclear extract from H9C2s were prepared as indicated in the Methods. Nuclear extracts were analyzed by WB for total CaMKII with selective antibody. CaMKII levels were averaged and normalized to histone 3 densitometry. * = p<0.05 vs Ctrl; # = p<0.05 vs PE. F: To evaluate nuclear ERK localization, the nuclear extracts were analyzed by WB for total ERK with specific antibody. ERK1/2 levels were normalized to histone 3 densitometry. *, P < 0.05 vs . Ctrl; #, P < 0.05 vs . PE. Data from all immunoblots were quantified by densitometric analysis. Each data point in all graphs represent the mean±SEM of 3 independent experiments.

Article Snippet: To investigate the effect of CaMKII inhibition on PE-mediated ERK activation, we pretreated H9C2 for 30 min. with the CaMKs inhibitor KN93 (5 μmol/L, methossibenensulphonamide, purchased from Seikagaku); alternatively we used of the selective CaMKII inhibitors AntCaNtide (10 μmol/L) [ , , ] and tat-CN17β (5 μmol/L) [ ].

Techniques: Inhibition, Activation Assay, Western Blot, Immunoprecipitation

Journal: PLoS ONE

Article Title: Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy

doi: 10.1371/journal.pone.0130477

Figure Lengend Snippet: A: H9C2 cells at ≈ 70% confluence were incubated 1 h at 37°C with 5 mL DMEM containing purified adenovirus at a multiplicity of infection (moi) of 100:1, encoding either the kinase-dead (CaMKII-DN, rCaMKIIdelta, K42M), or the wild type (CaMKII-WT, rCaMKIIdelta) variant of CaMKII or the empty virus as a negative control (Ctr). 48 h after the infection, the cells were stimulated with PE 100 nM for 24 h. Total RNA was isolated from H9C2s using TRIzol reagent, and cDNA was synthesized by means of a Thermo-Script RT-PCR System, following the manufacturer’s instruction. Then ANP gene expression was evaluated by real-time PCR. Results are expressed as mean±SEM from 3 independent experiments. The ratio of fold change was calculated using the Pfaffl method. * = p<0.05 vs Ctrl; # = p<0.05 vs PE. B: The H9C2s infected with adenoviruses encoding wilde type CaMKII (CaMKII-WT) and kinase dead CaMKII (CaMKII-DN) were stimulated with PE 100 nM for 24 h. Total cell lisates were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were corrected by Actin densitometry. Data from the immunoblots were quantified by densitometric analysis.* = p<0.05 vs Ctrl. Each data point in all graphs represents the mean±SEM of 3 independent experiments. C: H9C2 cells were pretreated with CaMK inhibitor KN93 (5 μmol/L), the selective inhibitors AntCaNtide (10 μmol/L) and tat-CN17β (10 μmol/L) and ERK specific inhibitor pathway UO126 (10 μmol/L) for 30 min. and then stimulated with PE (100 nmol/L) for 24 h. cDNA was synthesized from RNA obtained from H9C2s as indicated above. The ANF gene expression was evaluated by real-time PCR. Results are expressed as mean±SEM from 3 independent experiments. The ratio of fold change was calculated using the Pfaffl method. * = p<0.05 vs Ctrl; # = p<0.05 vs PE.

Article Snippet: To investigate the effect of CaMKII inhibition on PE-mediated ERK activation, we pretreated H9C2 for 30 min. with the CaMKs inhibitor KN93 (5 μmol/L, methossibenensulphonamide, purchased from Seikagaku); alternatively we used of the selective CaMKII inhibitors AntCaNtide (10 μmol/L) [ , , ] and tat-CN17β (5 μmol/L) [ ].

Techniques: Incubation, Purification, Infection, Variant Assay, Virus, Negative Control, Isolation, Synthesized, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell Reports Medicine

Article Title: Biguanides antithetically regulate tumor properties by the dose-dependent mitochondrial reprogramming-driven c-Src pathway

doi: 10.1016/j.xcrm.2025.101941

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-phospho-CaMKK2(Ser511) , Cell Signaling Technology , Cat12818S; RRID: AB_ 2798034.

Techniques: Virus, In Vivo, Recombinant, Protease Inhibitor, Red Blood Cell Lysis, Membrane, Bicinchoninic Acid Protein Assay, Staining, Software