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Procell Inc human lung cancer cell lines a549
Human Lung Cancer Cell Lines A549, supplied by Procell Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 86 stars, based on 1 article reviews

human lung cancer cell lines a549 - by Bioz Stars, 2026-05

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Expression of proteins associated with apoptosis and cell cycle regulation in NaAR-exposed A549 cells. ( a ) A549 cells were exposed to increasing concentrations of NaAR for 18 h, and morphological alterations were examined by phase-contrast microscopy. Nuclear staining was performed using Hoechst 33342, and fluorescence images were captured with Nikon Eclipse TE300 microscope (200×). ( b ) A549 and H1299 cells were treated with the indicated concentrations of NaAR for 18 h, followed by cell collection and lysis. Protein expression levels were analyzed by immunoblotting. ( c ) A549 cells were treated under the same conditions, and cell lysates were subjected to immunoblot analysis to evaluate target protein expression, β-actin used as a loading control. Arrows indicate non-apoptotic chromatin changes. Data represent results from at least three independent experiments. The arrows indicate large-scale segmented chromatin.

Journal: Cells

Article Title: Arsenic-Induced PPARγ, with the Coordinated Action of p62, Inhibits Apoptosis and Necroptosis and Activates the DNA Damage Response in A549 Lung Cancer Cells, Leading to Carcinogenesis

doi: 10.3390/cells15080659

Figure Lengend Snippet: Expression of proteins associated with apoptosis and cell cycle regulation in NaAR-exposed A549 cells. ( a ) A549 cells were exposed to increasing concentrations of NaAR for 18 h, and morphological alterations were examined by phase-contrast microscopy. Nuclear staining was performed using Hoechst 33342, and fluorescence images were captured with Nikon Eclipse TE300 microscope (200×). ( b ) A549 and H1299 cells were treated with the indicated concentrations of NaAR for 18 h, followed by cell collection and lysis. Protein expression levels were analyzed by immunoblotting. ( c ) A549 cells were treated under the same conditions, and cell lysates were subjected to immunoblot analysis to evaluate target protein expression, β-actin used as a loading control. Arrows indicate non-apoptotic chromatin changes. Data represent results from at least three independent experiments. The arrows indicate large-scale segmented chromatin.

Article Snippet: Human lung cancer cell lines A549 (CCL-185 TM ), H1299 (CRL-1803 TM ), and H460 (HTB-177 TM ) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Expressing, Microscopy, Staining, Fluorescence, Lysis, Western Blot, Control

NaAR-induced cell death and DNA damage is dependent on PARP-1 activation. ( a ) A549 cells were treated with increasing NaAR concentrations for 18 h or with 65 µM NaAR for up to 24 h. Target proteins in the cell lysates were then evaluated using immunoblotting. ( b ) Immunoblot of target proteins from A549 cells exposed to 65 µM NaAR for 18 h, with or without 50 µM NAD+ pretreatment for 2 h. ( c ) Hoechst 33342-stained cells. Lung cancer cells cultured on coverslips were treated with 65 µM NaAR for 18 h, with or without 50 µM NAD+ or 10 μM 3-AB pretreatment for 2 h, respectively. Scale bar: 25 µm. ( d ) Immunoblot of target proteins from A549 cells exposed to 65 µM NaAR for 18 h, with or without 10 μM 3-AB pretreatment for 2 h. β-Actin was used as the loading control.

Journal: Cells

doi: 10.3390/cells15080659

Figure Lengend Snippet: NaAR-induced cell death and DNA damage is dependent on PARP-1 activation. ( a ) A549 cells were treated with increasing NaAR concentrations for 18 h or with 65 µM NaAR for up to 24 h. Target proteins in the cell lysates were then evaluated using immunoblotting. ( b ) Immunoblot of target proteins from A549 cells exposed to 65 µM NaAR for 18 h, with or without 50 µM NAD+ pretreatment for 2 h. ( c ) Hoechst 33342-stained cells. Lung cancer cells cultured on coverslips were treated with 65 µM NaAR for 18 h, with or without 50 µM NAD+ or 10 μM 3-AB pretreatment for 2 h, respectively. Scale bar: 25 µm. ( d ) Immunoblot of target proteins from A549 cells exposed to 65 µM NaAR for 18 h, with or without 10 μM 3-AB pretreatment for 2 h. β-Actin was used as the loading control.

Article Snippet: Human lung cancer cell lines A549 (CCL-185 TM ), H1299 (CRL-1803 TM ), and H460 (HTB-177 TM ) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Activation Assay, Western Blot, Staining, Cell Culture, Control

NaAR exposure induces K63-linked RIP1-mediated NF-κB activation and MLKL downregulation. ( a , b ) Immunoblot of target proteins from A549 cells treated with increasing concentrations of NaAR for 18 h. ( c ) Immunoblot of target proteins from A549 cells exposed to 65 µM NaAR for 18 h, with or without 50 µM NAD + pretreatment for 2 h. ( d ) Immunoblot of target proteins from A549 cells exposed to 65 µM NaAR for 18 h, with or without 10 μM 3-AB pretreatment for 2 h. β-Actin was used as the loading control.

Journal: Cells

doi: 10.3390/cells15080659

Figure Lengend Snippet: NaAR exposure induces K63-linked RIP1-mediated NF-κB activation and MLKL downregulation. ( a , b ) Immunoblot of target proteins from A549 cells treated with increasing concentrations of NaAR for 18 h. ( c ) Immunoblot of target proteins from A549 cells exposed to 65 µM NaAR for 18 h, with or without 50 µM NAD + pretreatment for 2 h. ( d ) Immunoblot of target proteins from A549 cells exposed to 65 µM NaAR for 18 h, with or without 10 μM 3-AB pretreatment for 2 h. β-Actin was used as the loading control.

Article Snippet: Human lung cancer cell lines A549 (CCL-185 TM ), H1299 (CRL-1803 TM ), and H460 (HTB-177 TM ) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Activation Assay, Western Blot, Control

NaAR induces polyUb-PPARγ, and PPARγ knockdown leads to PARP-1 hyperactivation and necroptosis. ( a ) Immunoblot of PPARγ proteins from A549 cells treated with increasing NaAR concentrations for 18 h or with 65 µM NaAR for up to 24 h. ( b ) Immunoblot of target proteins from cells transfected with negative control (NC) or PPARγ siRNAs and then exposed to 65 µM NaAR for 18 h. ( c ) Cells were transfected with NC or PPARγ siRNAs and then exposed to NaAR as described in b, and the nuclei were stained with Hoechst 33342. Images were acquired with a fluorescence microscope. Scale bar: 25 µm. β-Actin was used as the loading control.

Journal: Cells

doi: 10.3390/cells15080659

Figure Lengend Snippet: NaAR induces polyUb-PPARγ, and PPARγ knockdown leads to PARP-1 hyperactivation and necroptosis. ( a ) Immunoblot of PPARγ proteins from A549 cells treated with increasing NaAR concentrations for 18 h or with 65 µM NaAR for up to 24 h. ( b ) Immunoblot of target proteins from cells transfected with negative control (NC) or PPARγ siRNAs and then exposed to 65 µM NaAR for 18 h. ( c ) Cells were transfected with NC or PPARγ siRNAs and then exposed to NaAR as described in b, and the nuclei were stained with Hoechst 33342. Images were acquired with a fluorescence microscope. Scale bar: 25 µm. β-Actin was used as the loading control.

Article Snippet: Human lung cancer cell lines A549 (CCL-185 TM ), H1299 (CRL-1803 TM ), and H460 (HTB-177 TM ) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Knockdown, Western Blot, Transfection, Negative Control, Staining, Fluorescence, Microscopy, Control

$The stability of PPARγ in NaAR-exposed A549 cells is regulated by proteasomes. ( a , b ) Immunoblot of target proteins from A549 cells exposed to 65 µM NaAR for 18 h, with 25 nM LMB or DMSO pretreatment for 2 h. ( c ) Immunoblot of target proteins from cells treated with 65 µM NaAR for 18 h in the presence of 25 nM LMB or 5 µM MG132 or both inhibitors. β-Actin was used as the loading control. ( d ) Immunoblot of target proteins from cells treated as described in ( a ) and ( b ) and subjected to subcellular fractionation into nucleus-rich, cytosolic, and particulate fractions. The purities of the nuclear, autophagosome, mitochondrial, and cytosolic fractions were determined by immunoblotting for HDAC1, LC3-II, SOD2, and β-actin, respectively (n = 3). ( e ) Cells on coverslips were treated as in ( a , b ), fixed, and stained with PPARγ (green) and p53 (red), followed by FITC- and rhodamine-conjugated secondary antibodies. The arrows indicate p53 in the cytosol. Nuclei were counterstained with Hoechst 33342 (blue) and images were acquired by fluorescence microscopy. Arrows indicate apoptotic nuclei. Scale bar: 25 µm.$

Journal: Cells

doi: 10.3390/cells15080659

Figure Lengend Snippet: The stability of PPARγ in NaAR-exposed A549 cells is regulated by proteasomes. ( a , b ) Immunoblot of target proteins from A549 cells exposed to 65 µM NaAR for 18 h, with 25 nM LMB or DMSO pretreatment for 2 h. ( c ) Immunoblot of target proteins from cells treated with 65 µM NaAR for 18 h in the presence of 25 nM LMB or 5 µM MG132 or both inhibitors. β-Actin was used as the loading control. ( d ) Immunoblot of target proteins from cells treated as described in ( a ) and ( b ) and subjected to subcellular fractionation into nucleus-rich, cytosolic, and particulate fractions. The purities of the nuclear, autophagosome, mitochondrial, and cytosolic fractions were determined by immunoblotting for HDAC1, LC3-II, SOD2, and β-actin, respectively (n = 3). ( e ) Cells on coverslips were treated as in ( a , b ), fixed, and stained with PPARγ (green) and p53 (red), followed by FITC- and rhodamine-conjugated secondary antibodies. The arrows indicate p53 in the cytosol. Nuclei were counterstained with Hoechst 33342 (blue) and images were acquired by fluorescence microscopy. Arrows indicate apoptotic nuclei. Scale bar: 25 µm.

Article Snippet: Human lung cancer cell lines A549 (CCL-185 TM ), H1299 (CRL-1803 TM ), and H460 (HTB-177 TM ) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Western Blot, Control, Fractionation, Staining, Fluorescence, Microscopy

p62 regulates p53 stability in NaAR-exposed A549 cells. ( a ) Immunoblot of p62 proteins from A549 cells treated with increasing concentrations of NaAR for 18 h or with 65 µM NaAR for up to 24 h. ( b , c ) Immunoblot of target proteins from cells transfected with negative control (NC) or p62 siRNAs and then exposed to 65 μM NaAR for 18 h. β-Actin was used as the loading control (n = 3).

Journal: Cells

doi: 10.3390/cells15080659

Figure Lengend Snippet: p62 regulates p53 stability in NaAR-exposed A549 cells. ( a ) Immunoblot of p62 proteins from A549 cells treated with increasing concentrations of NaAR for 18 h or with 65 µM NaAR for up to 24 h. ( b , c ) Immunoblot of target proteins from cells transfected with negative control (NC) or p62 siRNAs and then exposed to 65 μM NaAR for 18 h. β-Actin was used as the loading control (n = 3).

Article Snippet: Human lung cancer cell lines A549 (CCL-185 TM ), H1299 (CRL-1803 TM ), and H460 (HTB-177 TM ) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Western Blot, Transfection, Negative Control, Control

Subcellular localization of p62, p53, and PPARγ and their interactions in NaAR-exposed A549 cells. ( a ) Immunoblot of target proteins from cells treated with 65 μM NaAR for 18 h in the presence of either 25 nM LMB or DMSO. ( b , d ) Cells on coverslips were treated as in part ( a ), fixed, and stained with p62, p53, and PPARγ antibodies. Nuclei were counterstained with Hoechst 33342 (blue), and images were obtained by fluorescence microscopy. Scale bar: 25 µm. ( c ) Immunoblots of p62, p53, and PPARγ from cells treated with 65 µM NaAR for 12 h. After immunoblotting for p62 (input), 800 µg of the remaining protein was used for immunoprecipitation with p62 antibody, followed by immunoblotting for p62, p53, and PPARγ. β-Actin was used as the loading control (n = 3).

Journal: Cells

doi: 10.3390/cells15080659

Figure Lengend Snippet: Subcellular localization of p62, p53, and PPARγ and their interactions in NaAR-exposed A549 cells. ( a ) Immunoblot of target proteins from cells treated with 65 μM NaAR for 18 h in the presence of either 25 nM LMB or DMSO. ( b , d ) Cells on coverslips were treated as in part ( a ), fixed, and stained with p62, p53, and PPARγ antibodies. Nuclei were counterstained with Hoechst 33342 (blue), and images were obtained by fluorescence microscopy. Scale bar: 25 µm. ( c ) Immunoblots of p62, p53, and PPARγ from cells treated with 65 µM NaAR for 12 h. After immunoblotting for p62 (input), 800 µg of the remaining protein was used for immunoprecipitation with p62 antibody, followed by immunoblotting for p62, p53, and PPARγ. β-Actin was used as the loading control (n = 3).

Article Snippet: Human lung cancer cell lines A549 (CCL-185 TM ), H1299 (CRL-1803 TM ), and H460 (HTB-177 TM ) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Western Blot, Staining, Fluorescence, Microscopy, Immunoprecipitation, Control

Sodium arsenite (NaAR) induces polyubiquitination of PPARγ, which promotes DNA damage responses while suppressing apoptosis and necroptosis through NF-κB activation and MLKL downregulation. Disruption of PPARγ or modulation of PARP-1 activity shifts the balance toward necroptosis or apoptosis. p53 and p62 cooperate with PPARγ to regulate cell fate in A549 cells.

Journal: Cells

doi: 10.3390/cells15080659

Figure Lengend Snippet: Sodium arsenite (NaAR) induces polyubiquitination of PPARγ, which promotes DNA damage responses while suppressing apoptosis and necroptosis through NF-κB activation and MLKL downregulation. Disruption of PPARγ or modulation of PARP-1 activity shifts the balance toward necroptosis or apoptosis. p53 and p62 cooperate with PPARγ to regulate cell fate in A549 cells.

Article Snippet: Human lung cancer cell lines A549 (CCL-185 TM ), H1299 (CRL-1803 TM ), and H460 (HTB-177 TM ) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Activation Assay, Disruption, Activity Assay

GAK disruption induces stress fibre formation. (A) Transmission electron microscopy of wild-type (WT) A549 lung cancer cell line and the corresponding GAK-KO cells (×1000). Boxed regions are shown at high magnification (×5000). Yellow arrows indicate filamentous structures. Scale bars: 5 µm (×1000); 1 µm (×5000). (B) Immunofluorescence (IF) images of A549 WT and GAK-KO cells stained for myosin heavy chain (myosin IIa; green in merge), F-actin (phalloidin; magenta in merge) and DNA (DAPI; blue). Scale bars: 20 µm. (C) F-actin (phalloidin) staining in A549, non-targeting shRNA-transduced (A549/shNT), and GAK shRNA-transduced (A549/shGAK#1 and A549/shGAK#2) cells. Scale bars: 20 µm. (D) IB analysis of GAK expression in A549, A549/shNT, A549/shGAK#1, and A549/shGAK#2 cells. (E) F-actin (phalloidin) staining in A549 cells treated with or without gefitinib (30 µM, 3 h) treatment. Scale bars: 20 µm. (F) F-actin (phalloidin) staining in A549 WT, GAK-KO and GAK-KO cells transduced with an empty vector (GAK-KO/Empty) or GAK-WT (GAK-KO/GAK-WT). Scale bars: 20 µm. All panels show representative images from at least three independent experimental repeats.

Journal: Journal of Cell Science

Article Title: GAK antagonises ROCK-dependent regulation of actomyosin dynamics

doi: 10.1242/jcs.264117

Figure Lengend Snippet: GAK disruption induces stress fibre formation. (A) Transmission electron microscopy of wild-type (WT) A549 lung cancer cell line and the corresponding GAK-KO cells (×1000). Boxed regions are shown at high magnification (×5000). Yellow arrows indicate filamentous structures. Scale bars: 5 µm (×1000); 1 µm (×5000). (B) Immunofluorescence (IF) images of A549 WT and GAK-KO cells stained for myosin heavy chain (myosin IIa; green in merge), F-actin (phalloidin; magenta in merge) and DNA (DAPI; blue). Scale bars: 20 µm. (C) F-actin (phalloidin) staining in A549, non-targeting shRNA-transduced (A549/shNT), and GAK shRNA-transduced (A549/shGAK#1 and A549/shGAK#2) cells. Scale bars: 20 µm. (D) IB analysis of GAK expression in A549, A549/shNT, A549/shGAK#1, and A549/shGAK#2 cells. (E) F-actin (phalloidin) staining in A549 cells treated with or without gefitinib (30 µM, 3 h) treatment. Scale bars: 20 µm. (F) F-actin (phalloidin) staining in A549 WT, GAK-KO and GAK-KO cells transduced with an empty vector (GAK-KO/Empty) or GAK-WT (GAK-KO/GAK-WT). Scale bars: 20 µm. All panels show representative images from at least three independent experimental repeats.

Article Snippet: The human lung cancer cell line A549 (ATCC No. CCL-185), the human pancreatic cancer cell line PANC-1 (ATCC No. CRL-1469) and the human liver cancer cell line HepG2 (ATCC No. HB-8065) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in RPMI 1640 medium (Merck Sigma-Aldrich) supplemented with 10% heat-inactivated foetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin solution (Wako Pure Chemical) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Disruption, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, shRNA, Expressing, Transduction, Plasmid Preparation

GAK disruption promotes cell migration. (A) Representative phase-contrast images from a wound-healing assay of A549 WT, GAK-KO, GAK-KO/Empty, and GAK-KO/GAK-WT cells at 0, 12, 24 and 48 h after scratching. (B) Quantification of wound area after scratching over 48 h. Data are presented as mean±s.d. ( n =6). *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test). (C,D) Rose plots showing migration tracks of A549 WT (C) and GAK-KO (D) cells. Each line represents the track of an individual cell over 24 h. (E) Quantification of migration distance after 24 h. Data are presented as mean±s.d. ( n =10). *** P <0.001 (unpaired two-tailed Student's t -test).

Journal: Journal of Cell Science

Article Title: GAK antagonises ROCK-dependent regulation of actomyosin dynamics

doi: 10.1242/jcs.264117

Figure Lengend Snippet: GAK disruption promotes cell migration. (A) Representative phase-contrast images from a wound-healing assay of A549 WT, GAK-KO, GAK-KO/Empty, and GAK-KO/GAK-WT cells at 0, 12, 24 and 48 h after scratching. (B) Quantification of wound area after scratching over 48 h. Data are presented as mean±s.d. ( n =6). *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test). (C,D) Rose plots showing migration tracks of A549 WT (C) and GAK-KO (D) cells. Each line represents the track of an individual cell over 24 h. (E) Quantification of migration distance after 24 h. Data are presented as mean±s.d. ( n =10). *** P <0.001 (unpaired two-tailed Student's t -test).

Techniques: Disruption, Migration, Wound Healing Assay, Two Tailed Test

GAK disruption promotes MLC phosphorylation. (A) IB analysis of MLC (denoted MLC2 in the figure) and phosphorylated MLC2 in A549 WT and GAK-KO cells. Representative blots are shown. (B) Quantification of the relative band intensity of total MLC2, phosphorylated MLC2 (Ser19) and phosphorylated MLC2 (Thr18/Ser19), normalised to GAPDH or total MLC2. Data are presented as mean±s.d. ( n =4; unpaired two-tailed Student's t -test). (C) Immunoprecipitation (IP) using an anti-FLAG antibody was performed on lysates from 293T cells overexpressing FLAG-tagged GAK and V5-tagged MYPT1, MPRIP or the catalytic subunit of protein phosphatase 1 (PP1c), followed by immunoblotting (IB) with anti-V5 and anti-FLAG antibodies. In, Input (10%). Representative blots from at least three independent experimental repeats are shown.

Journal: Journal of Cell Science

Article Title: GAK antagonises ROCK-dependent regulation of actomyosin dynamics

doi: 10.1242/jcs.264117

Figure Lengend Snippet: GAK disruption promotes MLC phosphorylation. (A) IB analysis of MLC (denoted MLC2 in the figure) and phosphorylated MLC2 in A549 WT and GAK-KO cells. Representative blots are shown. (B) Quantification of the relative band intensity of total MLC2, phosphorylated MLC2 (Ser19) and phosphorylated MLC2 (Thr18/Ser19), normalised to GAPDH or total MLC2. Data are presented as mean±s.d. ( n =4; unpaired two-tailed Student's t -test). (C) Immunoprecipitation (IP) using an anti-FLAG antibody was performed on lysates from 293T cells overexpressing FLAG-tagged GAK and V5-tagged MYPT1, MPRIP or the catalytic subunit of protein phosphatase 1 (PP1c), followed by immunoblotting (IB) with anti-V5 and anti-FLAG antibodies. In, Input (10%). Representative blots from at least three independent experimental repeats are shown.

Techniques: Disruption, Phospho-proteomics, Two Tailed Test, Immunoprecipitation, Western Blot

Crosstalk with ROCK-dependent actomyosin regulation. (A) IF microscopy analysis of A549 WT and GAK-KO cells stained for myosin IIa (green in merge), F-actin (phalloidin; magenta in merge), and DNA (DAPI; blue). GAK-KO cells were treated with DMSO, Y-27632 (3–30 µM), or fasudil (30 µM) for 24 h. Scale bars: 20 µm. (B) IF microscopy analysis of A549 WT and GAK-KO cells stained for myosin IIa (green in merge), F-actin (phalloidin; magenta in merge), and DNA (DAPI; blue). GAK-KO cells were transduced with non-targeting shRNA (GAK-KO/shNT), ROCK1-targeted shRNA (GAK-KO/shROCK1#1 and GAK-KO/shROCK1#2), or ROCK2-targeted shRNA (GAK-KO/shROCK2). Scale bars: 20 µm. For A and B, representative images from at least three independent experimental repeats are shown. (C) IB analysis of ROCK1 and ROCK2 expression in A549 WT, GAK-KO, GAK-KO/shNT, GAK-KO/shROCK1#1, GAK-KO/shROCK1#2, and GAK-KO/shROCK2 cells. (D) Immunoprecipitation (IP) with anti-FLAG antibody was performed on lysates from 293T cells overexpressing FLAG-tagged GAK and V5-tagged RhoA, ROCK1 or ROCK2, followed by IB analysis with anti-V5 and anti-FLAG antibodies. For C and D, representative blots from at least three independent experimental repeats are shown. (E) Immunoprecipitation with anti-FLAG antibody was performed on lysates from 293T cells overexpressing FLAG-tagged GAK and V5-tagged Rho guanine nucleotide exchange factors (RhoGEFs) – ARHGEF1, ARHGEF2, ARHGEF7, ARHGEF9, ARHGEF11, or ARHGEF28 – followed by IB analysis with anti-V5 and anti-FLAG antibodies. (F) Quantification of the relative co-precipitation efficiency of ARHGEFs with GAK, with the co-precipitation efficiency of ARHGEF1 with GAK set to 1. Data are presented as mean±s.d. ( n =3). In, Input (10%).

Journal: Journal of Cell Science

Article Title: GAK antagonises ROCK-dependent regulation of actomyosin dynamics

doi: 10.1242/jcs.264117

Figure Lengend Snippet: Crosstalk with ROCK-dependent actomyosin regulation. (A) IF microscopy analysis of A549 WT and GAK-KO cells stained for myosin IIa (green in merge), F-actin (phalloidin; magenta in merge), and DNA (DAPI; blue). GAK-KO cells were treated with DMSO, Y-27632 (3–30 µM), or fasudil (30 µM) for 24 h. Scale bars: 20 µm. (B) IF microscopy analysis of A549 WT and GAK-KO cells stained for myosin IIa (green in merge), F-actin (phalloidin; magenta in merge), and DNA (DAPI; blue). GAK-KO cells were transduced with non-targeting shRNA (GAK-KO/shNT), ROCK1-targeted shRNA (GAK-KO/shROCK1#1 and GAK-KO/shROCK1#2), or ROCK2-targeted shRNA (GAK-KO/shROCK2). Scale bars: 20 µm. For A and B, representative images from at least three independent experimental repeats are shown. (C) IB analysis of ROCK1 and ROCK2 expression in A549 WT, GAK-KO, GAK-KO/shNT, GAK-KO/shROCK1#1, GAK-KO/shROCK1#2, and GAK-KO/shROCK2 cells. (D) Immunoprecipitation (IP) with anti-FLAG antibody was performed on lysates from 293T cells overexpressing FLAG-tagged GAK and V5-tagged RhoA, ROCK1 or ROCK2, followed by IB analysis with anti-V5 and anti-FLAG antibodies. For C and D, representative blots from at least three independent experimental repeats are shown. (E) Immunoprecipitation with anti-FLAG antibody was performed on lysates from 293T cells overexpressing FLAG-tagged GAK and V5-tagged Rho guanine nucleotide exchange factors (RhoGEFs) – ARHGEF1, ARHGEF2, ARHGEF7, ARHGEF9, ARHGEF11, or ARHGEF28 – followed by IB analysis with anti-V5 and anti-FLAG antibodies. (F) Quantification of the relative co-precipitation efficiency of ARHGEFs with GAK, with the co-precipitation efficiency of ARHGEF1 with GAK set to 1. Data are presented as mean±s.d. ( n =3). In, Input (10%).

Techniques: Microscopy, Staining, Transduction, shRNA, Expressing, Immunoprecipitation

Catalytic activity of GAK plays a limited role in actomyosin regulation. (A) Schematic of GAK and its mutants generated in this study. (B) F-actin (phalloidin) staining in A549 WT, GAK-KO, and GAK-KO cells transduced with an empty vector (Empty), GAK-WT, or GAK mutants (GAK-D173A, GAK-delM1, GAK-delM2, or GAK-delM3). Scale bars: 20 µm. (C–E) Quantification of mean phalloidin intensity (C), cell spread area (D) and aspect ratio (E). At least 60 cells per condition were quantified from three independent experiments and are presented in box plots. The box extends from the lower to the upper quartile; the middle line indicates the median; the cross indicates the mean; and the whiskers represent the minimum to maximum values, except for outliers. Data points outside 1.5× interquartile range from the quartiles are considered outliers and are shown as dots. *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test).

Journal: Journal of Cell Science

Article Title: GAK antagonises ROCK-dependent regulation of actomyosin dynamics

doi: 10.1242/jcs.264117

Figure Lengend Snippet: Catalytic activity of GAK plays a limited role in actomyosin regulation. (A) Schematic of GAK and its mutants generated in this study. (B) F-actin (phalloidin) staining in A549 WT, GAK-KO, and GAK-KO cells transduced with an empty vector (Empty), GAK-WT, or GAK mutants (GAK-D173A, GAK-delM1, GAK-delM2, or GAK-delM3). Scale bars: 20 µm. (C–E) Quantification of mean phalloidin intensity (C), cell spread area (D) and aspect ratio (E). At least 60 cells per condition were quantified from three independent experiments and are presented in box plots. The box extends from the lower to the upper quartile; the middle line indicates the median; the cross indicates the mean; and the whiskers represent the minimum to maximum values, except for outliers. Data points outside 1.5× interquartile range from the quartiles are considered outliers and are shown as dots. *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test).

Techniques: Activity Assay, Generated, Staining, Transduction, Plasmid Preparation

The IDR of GAK is involved in actomyosin regulation through interaction with ARHGEF2. (A) IB analysis of phosphorylated MLC (denoted MLC2 in the figure) (Thr18/Ser19) in A549 WT, GAK-KO and GAK-KO cells transduced with an empty vector (Empty), GAK-WT or GAK mutants (delM1, delM2 or delM3). (B) Quantification of the relative band intensity of phosphorylated MLC2 (Thr18/Ser19), normalised to GAPDH. Data are presented as mean±s.d. ( n =3; one-way ANOVA followed by Tukey–Kramer post hoc test). (C) Immunoprecipitation (IP) with anti-FLAG antibody was performed on lysates from 293T cells overexpressing FLAG-tagged GAK (WT or mutants) and V5-tagged PP1c, followed by IB analysis with anti-V5 and anti-FLAG antibodies. (D) Quantification of the relative co-precipitation efficiency of PP1c with GAK (WT or mutants). Data are presented as mean±s.d. ( n =4). *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test). (E) Immunoprecipitation with anti-FLAG antibody was performed on lysates from 293T cells overexpressing FLAG-tagged GAK (WT or mutants) and V5-tagged ARHGEF2, followed by IB analysis with anti-V5 and anti-FLAG antibodies. Inputs are 10% in C and E. (F) Quantification of the relative co-precipitation efficiency of ARHGEF2 with GAK (WT or mutants). Data are presented as mean±s.d. ( n =4; one-way ANOVA followed by Tukey–Kramer post hoc test). (G) IB analysis of ARHGEF2 and phosphorylated ARHGEF2 (Ser886) in A549 WT and GAK-KO cells. (H) Quantification of the relative band intensity of phosphorylated ARHGEF2, normalised to GAPDH. Data are presented as the mean±s.d. ( n =3; unpaired two-tailed Student's t -test). (I) F-actin (phalloidin) staining in A549 WT, GAK-KO and GAK-KO cells transduced with ARHGEF2-targeted shRNA (GAK-KO/shARHGEF2#1 and GAK-KO/shARHGEF2#2). Scale bars: 20 µm. (J–L) Quantification of phalloidin mean intensity (J), cell spread area (K) and cell aspect ratio (L). At least 30 cells per condition were quantified from three independent experiments and are presented in box plots. The box extends from the lower to the upper quartile; the middle line indicates the median; the cross indicates the mean; and the whiskers represent the minimum to maximum values, except for outliers. Data points outside 1.5× interquartile range from the quartiles are considered outliers and are shown as dots. *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test).

Journal: Journal of Cell Science

Article Title: GAK antagonises ROCK-dependent regulation of actomyosin dynamics

doi: 10.1242/jcs.264117

Figure Lengend Snippet: The IDR of GAK is involved in actomyosin regulation through interaction with ARHGEF2. (A) IB analysis of phosphorylated MLC (denoted MLC2 in the figure) (Thr18/Ser19) in A549 WT, GAK-KO and GAK-KO cells transduced with an empty vector (Empty), GAK-WT or GAK mutants (delM1, delM2 or delM3). (B) Quantification of the relative band intensity of phosphorylated MLC2 (Thr18/Ser19), normalised to GAPDH. Data are presented as mean±s.d. ( n =3; one-way ANOVA followed by Tukey–Kramer post hoc test). (C) Immunoprecipitation (IP) with anti-FLAG antibody was performed on lysates from 293T cells overexpressing FLAG-tagged GAK (WT or mutants) and V5-tagged PP1c, followed by IB analysis with anti-V5 and anti-FLAG antibodies. (D) Quantification of the relative co-precipitation efficiency of PP1c with GAK (WT or mutants). Data are presented as mean±s.d. ( n =4). *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test). (E) Immunoprecipitation with anti-FLAG antibody was performed on lysates from 293T cells overexpressing FLAG-tagged GAK (WT or mutants) and V5-tagged ARHGEF2, followed by IB analysis with anti-V5 and anti-FLAG antibodies. Inputs are 10% in C and E. (F) Quantification of the relative co-precipitation efficiency of ARHGEF2 with GAK (WT or mutants). Data are presented as mean±s.d. ( n =4; one-way ANOVA followed by Tukey–Kramer post hoc test). (G) IB analysis of ARHGEF2 and phosphorylated ARHGEF2 (Ser886) in A549 WT and GAK-KO cells. (H) Quantification of the relative band intensity of phosphorylated ARHGEF2, normalised to GAPDH. Data are presented as the mean±s.d. ( n =3; unpaired two-tailed Student's t -test). (I) F-actin (phalloidin) staining in A549 WT, GAK-KO and GAK-KO cells transduced with ARHGEF2-targeted shRNA (GAK-KO/shARHGEF2#1 and GAK-KO/shARHGEF2#2). Scale bars: 20 µm. (J–L) Quantification of phalloidin mean intensity (J), cell spread area (K) and cell aspect ratio (L). At least 30 cells per condition were quantified from three independent experiments and are presented in box plots. The box extends from the lower to the upper quartile; the middle line indicates the median; the cross indicates the mean; and the whiskers represent the minimum to maximum values, except for outliers. Data points outside 1.5× interquartile range from the quartiles are considered outliers and are shown as dots. *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test).

Techniques: Transduction, Plasmid Preparation, Immunoprecipitation, Two Tailed Test, Staining, shRNA

The IDR of GAK regulates actomyosin dynamics via the regulation of MLC gene expression. (A) RT-qPCR analysis of the relative mRNA expression levels of MYL9 in A549 WT and GAK-KO cells (clones 1-1, 1-2, and 2-1). GAPDH was used as an internal control. Data are presented as mean±s.d. ( n =4). *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test). (B) RT-qPCR analysis of relative mRNA expression levels of MYL9 in A549 WT, GAK-KO and GAK-KO cells transduced with an empty vector (Empty), GAK-WT, or GAK mutants (delM1, delM2 or delM3). GAPDH was used as an internal control. Data are presented as mean±s.d. ( n =4). *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test). (C) F-actin (phalloidin) staining in A549 WT, GAK-KO and GAK-KO cells transduced with non-targeting shRNA (A549/shNT) or MYL9-targeted shRNA (GAK-KO/shMYL9#1, GAK-KO/shMYL9#2, and GAK-KO/shMYL9#3). Scale bars: 20 µm. (D) Quantification of phalloidin mean intensity. At least 30 cells per condition were quantified from three independent experiments and are presented in box plots. The box extends from the lower to the upper quartile; the middle line indicates the median; the cross indicates the mean; and the whiskers represent the minimum to maximum values, except for outliers. Data points outside 1.5× interquartile range from the quartiles are considered outliers and are shown as dots. *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test).

Journal: Journal of Cell Science

Article Title: GAK antagonises ROCK-dependent regulation of actomyosin dynamics

doi: 10.1242/jcs.264117

Figure Lengend Snippet: The IDR of GAK regulates actomyosin dynamics via the regulation of MLC gene expression. (A) RT-qPCR analysis of the relative mRNA expression levels of MYL9 in A549 WT and GAK-KO cells (clones 1-1, 1-2, and 2-1). GAPDH was used as an internal control. Data are presented as mean±s.d. ( n =4). *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test). (B) RT-qPCR analysis of relative mRNA expression levels of MYL9 in A549 WT, GAK-KO and GAK-KO cells transduced with an empty vector (Empty), GAK-WT, or GAK mutants (delM1, delM2 or delM3). GAPDH was used as an internal control. Data are presented as mean±s.d. ( n =4). *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test). (C) F-actin (phalloidin) staining in A549 WT, GAK-KO and GAK-KO cells transduced with non-targeting shRNA (A549/shNT) or MYL9-targeted shRNA (GAK-KO/shMYL9#1, GAK-KO/shMYL9#2, and GAK-KO/shMYL9#3). Scale bars: 20 µm. (D) Quantification of phalloidin mean intensity. At least 30 cells per condition were quantified from three independent experiments and are presented in box plots. The box extends from the lower to the upper quartile; the middle line indicates the median; the cross indicates the mean; and the whiskers represent the minimum to maximum values, except for outliers. Data points outside 1.5× interquartile range from the quartiles are considered outliers and are shown as dots. *** P <0.001 (one-way ANOVA followed by Tukey–Kramer post hoc test).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Clone Assay, Control, Transduction, Plasmid Preparation, Staining, shRNA

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