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human lung cancer cell lines a549 (ATCC)

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ATCC human lung cancer cell lines a549
Human Lung Cancer Cell Lines A549, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 9098 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+lung+cancer+cell+lines+a549/A549/pm42152077-65-16-23
Average 99 stars, based on 9098 article reviews

human lung cancer cell lines a549 - by Bioz Stars, 2026-10

99/100 stars

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Multiple Displacement Amplification:

Article Title: Mitofusin-2 suppresses tumor immune escape through EGFR/STAT3-mediated PD-L1 transcription
Article Snippet: .. The human lung cancer cell lines A549 (CCL-185, RRID:CVCL_0023), NCI-H1299(CRL-5803, RRID:CVCL_0060), HCC827(CRL-2868, RRID:CVCL_2063), NCI-H1703(CRL-5889,RRID:CVCL_1490) and NCI-H1975 (CRL-5908, RRID:CVCL_1511), the human kidney cell line 786-O(CRL-1932, RRID:CVCL_1051), 769-P(CRL-1933, RRID:CVCL_1050) and the human breast cancer cell line MDA-MB-231(CRM-HTB-26, RRID:CVCL_0062) were purchased from ATCC (USA). .. OS-RC-2(CL-0177, RRID:CVCL_1626) and PC-9(CL-0668, RRID:CVCL_B260) were purchased from Procell (Wuhan, China), the mouse lung cancer cell line LL/2 (LLC1)(C5094, RRID: CVCL_4358) and the mouse kidney cancer cell line RENCA (C5190; RRID:CVCL_2174) were purchased from the BDBIO (Hangzhou, China).

Article Title: Mitofusin-2 suppresses tumor immune escape through EGFR/STAT3-mediated PD-L1 transcription.
Article Snippet: .. Cell lines The human lung cancer cell lines A549 (CCL-185, RRID:CVCL_0023), NCI-H1299(CRL-5803, RRID:CVCL_0060), HCC827(CRL-2868, RRID:CVCL_2063), NCI-H1703( CRL5889,RRID:CVCL_1490) and NCI-H1975 (CRL-5908, RRID:CVCL_1511), the human kidney cell line 786-O(CRL-1932, RRID:CVCL_1051), 769-P(CRL-1933, RRID:CVCL_1050) and the human breast cancer cell line MDA-MB-231(CRM-HTB-26, RRID:CVCL_0062) were purchased from ATCC (USA). .. OS-RC-2( CL-0177, RRID:CVCL_1626) and PC-9(CL-0668, RRID:CVCL_B260) were purchased from Procell (Wuhan China), the mouse lung cancer cell line LL/2 (LLC1)(C5094, RRID: CVCL_4358) and the mouse kidney cancer cell line RENCA (C5190; RRID:CVCL_2174) were purchased from the BDBIO (Hangzhou, China).

Cell Culture:

Article Title: Mitotic catastrophe-related six-gene signature predicts prognosis, tumor immune landscape, and therapeutic response in lung adenocarcinoma.
Article Snippet: .. Cell culture and transfection Human normal bronchial epithelial cells BEAS-2B (CRL-3588, ATCC, Manassas, VA, USA) and human lung cancer cell lines A549 (CRM-CCL-185, ATCC, Manassas, VA, USA) and NCI-H838 (CRL5844, ATCC, Manassas, VA, USA) were used. .. Cells were cultured in DMEM (PM150210, Procell, Wuhan, China) supplemented with 10% fetal bovine serum (FBS) (164210, Procell) and 1% penicillin– streptomycin (Procell) under standard conditions (37 °C, 5% CO2, saturated humidity) in a tri-gas incubator (Thermo Fisher Scientific, Waltham, MA, USA).

Transfection:

Modification:

Article Title: MOB2 Loss Sensitizes Lung Cancer Cells to PARP Inhibition Through p53-Dependent DNA Damage Signaling.
Article Snippet: .. The human lung cancer cell lines A549 (ATCC, CCL-185, CVCL-0023) and H1299 (ATCC, CRL-5803, CVCL-0060) were kindly provided by Dr. Alexander Hergovich (Evotec, Toulouse, France) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; https://doi.org/10.3390/cimb48030295 Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA). ..

Image Search Results

Internalization of SD-EVLPs and its inhibitory effects on lung cancer cell proliferation (A) Internalization of SD-EVLPs by lung cancer cells; (B, C) CCK-8 assays assessing the effects of SD-EVLPs on lung cancer cell viability; (D–G) EdU assays evaluating the proliferation of A549 and NCI-H1299 cells; (H–I) Colony formation assays assessing the proliferative capacity of A549 and NCI-H1299 cells. Data are presented as the mean ± SEM, n = 3 independent experiments. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Journal: Frontiers in Oncology

Article Title: Selaginella doederleinii -derived extracellular vesicle-like particles suppress lung cancer with ferroptosis-associated changes and modulation of the FABP4/PPARG/GPX4 axis

doi: 10.3389/fonc.2026.1829211

Figure Lengend Snippet: Internalization of SD-EVLPs and its inhibitory effects on lung cancer cell proliferation (A) Internalization of SD-EVLPs by lung cancer cells; (B, C) CCK-8 assays assessing the effects of SD-EVLPs on lung cancer cell viability; (D–G) EdU assays evaluating the proliferation of A549 and NCI-H1299 cells; (H–I) Colony formation assays assessing the proliferative capacity of A549 and NCI-H1299 cells. Data are presented as the mean ± SEM, n = 3 independent experiments. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Human lung cancer cell lines A549 (Cat. No.: CL-0016) and NCI-H1299 (Cat. No.: CL-0165) were purchased from Wuhan Procell Life Science & Technology Co., Ltd. Four-week-old male BALB/c nude mice (SPF grade) were purchased from Hunan Silaike Jingda Laboratory Animal Co., Ltd. (Changsha, China), with the Animal Production License No.: SCXK (Xiang) 2021-0002.

Techniques: CCK-8 Assay, Control

SD-EVLPs inhibit migration and invasion of lung cancer cells (A–D) Wound healing assays assessing the effect of SD-EVLPs on the migration of A549 and NCI-H1299 cells; (E, F) Transwell assays evaluating the invasion ability of A549 and NCI-H1299 cells; (G–I) Western blot analysis of E-cadherin and N-cadherin protein expression. Data are presented as the mean ± SEM, n = 3 independent experiments. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Journal: Frontiers in Oncology

Article Title: Selaginella doederleinii -derived extracellular vesicle-like particles suppress lung cancer with ferroptosis-associated changes and modulation of the FABP4/PPARG/GPX4 axis

doi: 10.3389/fonc.2026.1829211

Figure Lengend Snippet: SD-EVLPs inhibit migration and invasion of lung cancer cells (A–D) Wound healing assays assessing the effect of SD-EVLPs on the migration of A549 and NCI-H1299 cells; (E, F) Transwell assays evaluating the invasion ability of A549 and NCI-H1299 cells; (G–I) Western blot analysis of E-cadherin and N-cadherin protein expression. Data are presented as the mean ± SEM, n = 3 independent experiments. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Techniques: Migration, Western Blot, Expressing, Control

RT-qPCR and Western blot analysis of ferroptosis-related gene expression in lung cancer cells (A, B) RT-qPCR analysis of GPX4 and SLC7A11 mRNA expression in A549 cells; (C, D) RT-qPCR analysis of GPX4 and SLC7A11 mRNA expression in NCI-H1299 cells; (E–H) Western blot analysis of GPX4 and xCT protein expression in lung cancer cells; (I) TEM assessment of mitochondrial ultrastructure. Data are presented as the mean ± SEM, n = 3 independent experiments. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Journal: Frontiers in Oncology

Article Title: Selaginella doederleinii -derived extracellular vesicle-like particles suppress lung cancer with ferroptosis-associated changes and modulation of the FABP4/PPARG/GPX4 axis

doi: 10.3389/fonc.2026.1829211

Figure Lengend Snippet: RT-qPCR and Western blot analysis of ferroptosis-related gene expression in lung cancer cells (A, B) RT-qPCR analysis of GPX4 and SLC7A11 mRNA expression in A549 cells; (C, D) RT-qPCR analysis of GPX4 and SLC7A11 mRNA expression in NCI-H1299 cells; (E–H) Western blot analysis of GPX4 and xCT protein expression in lung cancer cells; (I) TEM assessment of mitochondrial ultrastructure. Data are presented as the mean ± SEM, n = 3 independent experiments. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Techniques: Quantitative RT-PCR, Western Blot, Gene Expression, Expressing, Control

SD-EVLPs modulate the FABP4/PPARG/GPX4-associated pathway in lung cancer cells (A–D) RT-qPCR analysis of FABP4 and PPARG mRNA expression in A549 and NCI-H1299 cells; (E–H) Western blot analysis of FABP4 and PPARG protein expression in A549 and NCI-H1299 cells. Data are presented as the mean ± SEM, n = 3 independent experiments. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Journal: Frontiers in Oncology

Article Title: Selaginella doederleinii -derived extracellular vesicle-like particles suppress lung cancer with ferroptosis-associated changes and modulation of the FABP4/PPARG/GPX4 axis

doi: 10.3389/fonc.2026.1829211

Figure Lengend Snippet: SD-EVLPs modulate the FABP4/PPARG/GPX4-associated pathway in lung cancer cells (A–D) RT-qPCR analysis of FABP4 and PPARG mRNA expression in A549 and NCI-H1299 cells; (E–H) Western blot analysis of FABP4 and PPARG protein expression in A549 and NCI-H1299 cells. Data are presented as the mean ± SEM, n = 3 independent experiments. Compared with the control group, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control

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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