Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Effect of JNK inhibition on A549 cell viability as a function of cisplatin concentration. (a, b) 7.5 μg/mL concentration of cisplatin becomes lethal in combination with JNK inhibitor SP600125. However, at higher cisplatin concentrations, the inhibitor either protects against cisplatin-induced cell death (a) or does not have any effect (b). (c) Similar effects are observed in the colon cancer cell line DLD-1. (d–g) SP600125 potentiates the appearance of cells with apoptotic morphology in 7.5 μg/mL cisplatin-treated A549 cells (d, e), in contrast to 30 μg/mL cisplatin-treated A549 cells (f, g). Relative cell viability is cell viability after 72 h treatment normalized by initial viability (measured by the MTT method). Representative test results (all measurements were performed in quadruplicate) from more than five experiments are presented. Fluorescent pictures were obtained with a mixture of AO/EB dyes as described in the section. SP—JNK inhibitor SP600125 (20 μM).

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Inhibition, Concentration Assay

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: JNK inhibitor SP600125 reduces JNK target transcription factor c-Jun phosphorylation in cisplatin-treated A549 cells. Different concentrations of cisplatin were used. (a) Representative Western blots from 20 and 40 h of treatments are shown. Total protein Coomassie-stained polyacrylamide gels serve as loading controls. (b) Repeated addition (after 24 h) of SP potentiates suppression of c-Jun phosphorylation in response to cisplatin treatment. (c) Repeated addition of SP strengthens the protective effect of JNK inhibition at 15−30 μg/mL concentrations of cisplatin in A549 cells. SP—JNK inhibitor SP600125 (20 μM). p *** < 0.0005, N = 3.

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Phospho-proteomics, Western Blot, Staining, Inhibition

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Cisplatin concentration-dependent effect of different JNK inhibitors on A549 cell viability. Different JNK inhibitors show the same dependence on cisplatin concentration on cell viability. Statistically significant reduction in cell viability is observed at 7.5 μg/mL cisplatin in combination with 10 μM AS601245 (a), 10 μM bentamapimod (b), 2 μM JNK inhibitor IX (c), and 5 μM XG-102 (d). Representative test results (all measurements were performed in quadruplicate) from more than five experiments are presented. (e) Reduction in c-Jun phosphorylation upon the addition of JNK inhibitor XG-102, as determined by Western blot. Representative Western blots are shown. Total protein Coomassie-stained polyacrylamide gels serve as loading controls.

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Concentration Assay, Phospho-proteomics, Western Blot, Staining

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Activation of TP53 in A549 cells in response to different concentrations of cisplatin. (a, b) Expression and phosphorylation at serine-6 is induced by cisplatin and is maximal at 15 μg/mL (6 h of cisplatin treatment). (c, d) Prolonged and increasing expression and phosphorylation of TP53 in cells treated with either 7.5 μg/mL or 30 μg/mL of cisplatin. (e) TP53 activator nutlin-3a potentiates cisplatin-induced decrease in cell viability. (f) Nutlin-3a reduces viability of cisplatin + SP600125-treated cells. C—control without cisplatin; Nut—nutlin-3a (10 μM); SP—SP600125 (20 μM). Representative test results (all measurements were performed in quadruplicate) from more than five experiments are presented, p ** < 0.005, p *** < 0.0005, N = 4. (g) Nutlin induces expression of TP53. Representative Western blots are shown. Total protein Coomassie-stained polyacrylamide gels serve as loading controls.

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Activation Assay, Expressing, Phospho-proteomics, Control, Western Blot, Staining

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Involvement of AKT signaling pathway in cisplatin-induced A549 cell death. (a) AKT protein level does not depend on cisplatin concentration. 6-h-long exposure is presented in the Western blot picture. (b) Phosphorylation/activation of AKT is cisplatin concentration-dependent. 6-h-long exposure is presented in the Western blot picture. (c) AKT protein level does not change during 40 h of cisplatin treatment. (d) Dynamics of AKT activation following cisplatin (7.5 or 30 μg/mL) treatment. (e, f) AKT inhibitor capivasertib enhances cell death at all concentrations of cisplatin used both in the absence (e) and presence (f) of JNK inhibitor SP600125. (g, h) GSK3-β inhibitor SB415286 protects A549 cells from cisplatin both in the absence (g) and presence (h) of JNK inhibition. Representative test results (all measurements were performed in quadruplicate) from more than five experiments are presented. p*<0.05, p**<0.005, p***<0.0005, N = 4. (i) Capivasertib inhibits AKT activity as shown by the inhibition of AKT molecular target GSK3-β phosphorylation at serine-9. Representative Western blots are shown. Total protein Coomassie-stained polyacrylamide gels serve as loading controls. C—control without cisplatin; CAP—capivasertib (10 μM); SP—SP600125 (20 μM), SB—GSK3 inhibitor SB415286 (15 μM).

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Concentration Assay, Western Blot, Phospho-proteomics, Activation Assay, Inhibition, Activity Assay, Staining, Control

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Opposite changes in TP53 and AKT phosphorylation following SP600125 treatment of A549 cells exposed to different concentrations of cisplatin. (a) Expression of TP53 is increased in response to the combination of SP + 7.5 μg/mL cisplatin, in contrast to the combination of SP + 30 μg/mL cisplatin. (b) Phosphorylation of TP53 (serine-6) is increased in response to the combination of SP + 7.5 μg/mL cisplatin, in contrast to the combination of SP + 30 μg/mL cisplatin. (c) Phosphorylation of AKT (threonine-308) is decreased in response to the combination of SP + 7.5 μg/mL cisplatin, in contrast to the combination of SP + 30 μg/mL cisplatin. Representative Western blots are shown. Total protein Coomassie-stained polyacrylamide gels serve as a loading control. SP—JNK inhibitor SP600125 (20 μM); 20 h of treatment.

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Phospho-proteomics, Expressing, Western Blot, Staining, Control

Journal: iScience

Article Title: Targeted delivery of immune-stimulating bispecific RNA, inducing apoptosis and anti-tumor immunity in cancer cells

doi: 10.1016/j.isci.2024.109068

Figure Lengend Snippet:

Article Snippet: A549-Dual RIG-I KO , InvivoGen , a549d-korigi.

Techniques: Isolation, Recombinant, WST-1 Assay, Screening, Control, Software

Journal: Molecular Pharmaceutics

Article Title: Fabrication of RIG-I-Activating Nanoparticles for Intratumoral Immunotherapy via Flash Nanoprecipitation

doi: 10.1021/acs.molpharmaceut.5c00125

Figure Lengend Snippet: RANs are endosomolytic, immunostimulatory in vitro, and stable in serum. (A) Experimental schematic of Gal8-YFP assay used to measure endosomolytic activity of RANs and representative microscopy images of MDA-MB-231 Gal8-YFP cells treated with a RAN formulation and a PBS control. (B) Dose response curves of MDA-MB-231 Gal8-YFP cells treated with indicated RAN formulations ( n = 3 biological replicates per group). All RANs promoted endosomolytic activity in a dose-dependent manner as indicated by (C) calculated EC 50 values of each RAN formulation. P values determined by an ordinary one-way ANOVA test with Tukey’s test for multiple comparisons. (D) Dose response curves for relative IFN-I production by A549-Dual reporter cells treated with RAN formulations, micelleplex, and free SLR14 controls ( n = 3 biological replicates per group). (E) Calculated EC 50 values for relative IFN-I production. P values determined by an ordinary one-way ANOVA test with Dunnett’s test for multiple comparisons. (F) Dose response curves for IFN-β secretion by isolated bone marrow-derived macrophages (BMDMs) treated with RAN formulations ( n = 2 biological replicates per group). (G) Calculated EC 50 values and (H) maximum secreted IFN-β levels. P values determined by an ordinary one-way ANOVA test with Tukey’s test for multiple comparisons. (I) Serum stability analysis of RAN 2 kDa (green), RAN 10 kDa (yellow), and micelleplex (red) immunostimulatory activity in A549-Dual IFN-I reporter cells after 24 h incubation in either 50% FBS or PBS at 37 °C. Replicates are biological, and data are shown as mean ± SD * signifies P < 0.05, ** signifies P < 0.01, *** signifies P < 0.001, **** signifies P < 0.0001.

Article Snippet: A549-Dual human lung adenocarcinoma cells (InvivoGen) were cultured in Dulbecco’s modified MEM (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, Gibco), 100 U/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), 2 mM l -glutamine, 25 mM HEPES (Invitrogen), 4.5 g/L d -glucose, and 100 μg/mL Normocin (InvivoGen).

Techniques: In Vitro, Activity Assay, Microscopy, Formulation, Control, Isolation, Derivative Assay, Stability, Analysis, Incubation

Journal: ACS Omega

Article Title: Enzymatic Glucosylation Enhances the Solubility of Niclosamide but Abrogates Its Therapeutic Efficacy

doi: 10.1021/acsomega.5c12185

Figure Lengend Snippet: Antiviral and cytotoxicity properties of Nic and Nic-Glc against SARS-CoV-2 in (A) Vero E6 cells, (B) A549-hACE2 cells, and (C) Huh7.5 cells. Percent viral infectivity is shown on left y -axis and percent cell viability is shown on the right y -axis (mean ± SD; n = 4). All values are normalized to the nontreated controls. We also confirmed that Remdesivir showed activity as expected and that DMSO alone had no significant antiviral activity ( Figures S3 and S4 , Table S1 ).

Article Snippet: For SARS-CoV-2 experiments in Vero E6 and A549-hACE2 cells (InvivoGen), we used the previously described SARS-CoV-2/human/Denmark/DK-AHH1/2020 isolate (GenBank accession no. MZ049597 ).

Techniques: Infection, Activity Assay

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: Characterization of A549 and A549/DDP cells. (A) A549 and A549/DDP cells were exposed to different concentrations of DDP (1–32 µg/ml) for 24 h, and cell viability was determined by Cell Counting Kit-8 assay. The protein expression levels of (B) E-cadherin, vimentin, α-SMA and (C) CIP2A, p-AKT, AKT, p-mTOR were measured using western blotting; E, epithelial; SMA, smooth muscle actin; p-mTOR, phosphorylated mammalian target of rapamycin; AKT, protein kinase B; DDP, cisplatin; CIP2A, cancerous inhibitor of protein phosphatase 2A; IC 50 , half maximal inhibitory concentration.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Cell Counting, Expressing, Western Blot, Concentration Assay

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII induce cytotoxicity in A549 and A549/DDP cells. (A) Chemical structure of PPI and PPVII. (B) A549 and A549/DDP cells were treated with different dose of PPI (0.5–8 µg/ml) and PPVII (0.5–8 µg/ml) for 24 h, respectively. In A549 cells, the cell inhibition rate of 0.5 µg/ml PPI was significantly different from other concentration groups (**P<0.01); 1.0 µg/ml was significantly different from 4.0 and 8.0 µg/ml (**P<0.01), and 2.0 µg/ml was significantly different from 8.0 µg/ml (**P<0.01). As for A549/DDP cells, the cell inhibition rates of 0.5 µg/ml PPI and 1.0 µg/ml were both significantly different from other concentration groups ( ## P<0.01), and 2.0 µg/ml was significantly different from 8.0 µg/ml ( ## P<0.01). In A549 cells, there were significant differences in cell inhibition rates of PPVII among different concentration groups (**P<0.01); as for A549/DDP cells, there were significant differences among different concentration groups except for 4.0 and 8.0 µg/ml ( ## P<0.01). PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Inhibition, Concentration Assay

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII possess chemo-sensitizing effects on A549/DDP cells. (A) A549/DDP cells treated with indicated doses of DDP, or DDP plus either 0.375 µg/ml PPI or 0.75 µg/ml PPI, respectively for 24 h. (B) A549/DDP cells treated with indicated doses of DDP, or DDP plus either 0.375 µg/ml PPVII or 0.75 µg/ml PPVII, respectively for 24 h, cell viability was measured with Cell Counting Kit-8 assay. Data were presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 vs. DDP treated group. # P<0.05 and ## P<0.01. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Cell Counting, Standard Deviation

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII reduce the IC 50 value of cisplatin in A549/DDP cells

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques:

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII enhance DDP-induced apoptosis. A549/DDP cells were treated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, respectively. Apoptosis of cells was detected by Annexin V-FITC/PI staining and flow cytometry. The apoptotic percentage is shown as a bar graph. Data represents the mean ± standard deviation of three independent experiments. **P<0.01 and ***P<0.001. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Staining, Flow Cytometry, Standard Deviation

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII enhance DDP-induced apoptosis through the P53 pathway and caspases-dependent pathway. A549/DDP cells were treated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, respectively. The protein expression levels of P53, Bax, Bcl-2 (A), PARP, C-PARP, pro-Caspase-3, C-Caspase-3 (B) were measured using western blotting. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; C-PARP, cleaved-poly (ADP-ribose) polymerase 1.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Expressing, Western Blot

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII reverse EMT and suppress the CIP2A/AKT/mTOR pathway. A549/DDP cells were incubated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, then cells were harvested for further western blotting analysis. (A) The protein expression levels of E-cadherin, vimentin, α-SMA in A549/DDP cells were measured. (B) The protein expression levels of CIP2A, p-AKT, AKT, p-mTOR, m-TOR were measured. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; p-AKT, phosphorylated-protein kinase B; mTOR, mammalian target of rapamycin; CIP2A, cancerous inhibitor of protein phosphatase 2A; SMA, smooth muscle actin; E, epithelial.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Incubation, Western Blot, Expressing

Journal: Nature Communications

Article Title: Kindlin-2 links mechano-environment to proline synthesis and tumor growth

doi: 10.1038/s41467-019-08772-3

Figure Lengend Snippet: Kindlin-2 interacts with PYCR1 and colocalizes with PYCR1 in mitochondria. a , b Co-IP of PYCR1 with kindlin-2. Human A549 ( a ) or HCI-H358 ( b ) cells were analyzed by IP with monoclonal anti-kindlin-2 antibody or irrelevant mouse IgG (as a control) as described in the Methods. The cell lysates (lane 1), control IgG (lane 2), and anti-kindlin-2 immunoprecipitates (lane 3) were analyzed by western blotting with antibodies as indicated. c Co-IP of PYCR1 with FLAG-kindlin-2. Kindlin-2 KO A549 cells were infected with lentiviral vector encoding 3xFLAG-tagged kindlin-2 (3fl-K2) or control lentiviral vector lacking kindlin-2 sequence (3fl). The 3fl-K2 and control 3fl infectants were analyzed by IP with anti-FLAG antibody. The 3fl-K2 cell lysates (lane 1) and IP samples from 3fl (lane 2) or 3fl-K2 (lane 3) cells were analyzed by western blotting with kindlin-2 or PYCR1 antibodies. d GST-kindlin-2 bound to glutathione-Sepharose beads were incubated with increased concentrations (lane 2, 0 μg ml − 1 ; lane 3, 10 μg ml − 1 ; lane 4, 20 μg ml − 1 ; lane 5, 40 μg ml − 1 ) of purified His-tagged PYCR1. GST-kindlin-2 fusion protein pulldown was analyzed as described in the Methods. The sample in lane 1 was prepared as that in lane 4, except GST-kindlin-2 was replaced with GST. e – g GST-tagged full-length or mutant forms of kindlin-2 (illustrated in e ) or GST alone (as a negative control) were used to pull down His-tagged PYCR1 as described in the Methods. The inputs, GST and GST-kindlin-2 fusion protein pulldowns, were analyzed by western blotting with antibodies against His and GST, respectively. h A549 cells were plated on fibronectin-coated coverslips and dually stained with mouse monoclonal anti-kindlin-2 and rabbit polyclonal anti-PYCR1 antibodies. The primary antibodies were detected with Alexa Fluor 488-conjugated anti-mouse IgG or Alexa Fluor 647-conjugated anti-rabbit IgG secondary antibodies. Scale bar = 10 μm. i The cytosolic fraction (Cyto, lane 1), mitochondrial fraction (Mito, lane 2), and total cell lysates (Total, lane 3) were analyzed by western blotting with antibodies to kindlin-2, PYCR1, tubulin, and prohibitin-2 (PHB2) as indicated

Article Snippet: Briefly, A549 cell lysates were immunoprecipitated with mouse anti-kindlin-2 antibody (clone 3A3.5 ) or control mouse IgG (Santa Cruz, Cat# sc-2025).

Techniques: Co-Immunoprecipitation Assay, Control, Western Blot, Infection, Plasmid Preparation, Sequencing, Incubation, Purification, Mutagenesis, Negative Control, Staining

Journal: Nature Communications

Article Title: Kindlin-2 links mechano-environment to proline synthesis and tumor growth

doi: 10.1038/s41467-019-08772-3

Figure Lengend Snippet: Formation of the kindlin-2-PYCR1 complex in cells. a – e Kindlin-2 KO A549 cells were infected with lentiviral vector encoding mcherry-tag kindlin-2 (mCh-K2) or mcherry vector lacking kindlin-2 sequence (mCh). Three days later, A549 cells (lane 1), kindlin-2 KO cells (lane 2), mCh infectants (lane 3), and mCh-K2 infectants (lane 4) were analyzed by western blotting with antibodies recognizing kindlin-2 and GAPDH ( d ). Kindlin-2 KO cells expressing mcherry-kindin-2 were stained with anti-PYCR1 antibody and DAPI, and analyzed by confocal microscopy. The maximum-intensity projection views of a representative A549 mCh-K2 cell reconstructed from Z-series images taken at various heights ( a ). Extended focal planes of focal adhesions show that mcherry-kindlin-2 is concentrated in focal adhesions, whereas there is virtually no PYCR1 in focal adhesions (open arrows) ( b ). Image layers taken above focal adhesions show that a fraction of mcherry-kindlin-2 is colocalized with PYCR1 in the mitochondria (solid arrows) ( c ). Fluorescence images of mcherry-kindlin-2 (red), PYCR1 (green), and merged images with DAPI (blue) are shown in the left, middle, and right column, respectively. Scale bar = 15 μm. High-magnification images of mcherry-kindlin-2 (red), PYCR1 (green), and DAPI (blue) taken with focal plane above focal adhesions ( e ). Scale bar = 7.5 μm. f Co-IP of PYCR1 with kindlin-2 from mitochondrial fraction. Cytosolic fraction (lane 1), mitochondrial fraction (lane 2), and total lysates (lane 3) from A549 cells were analyzed by IP with anti-kindlin-2 antibody (lanes 1–3) or irrelevant mouse IgG (lanes 4–6). The samples were analyzed by western blotting with antibodies to kindlin-2 and PYCR1. g , h FLIM-FRET analyses. Kindlin-2 KO cells were transfected with vectors encoding mClover-kindin-2 or mClover-F1 and F3 deletion mutant of kindlin-2 and mRuby-PYCR1, and analyzed by FLIM. The fluorescence images of mClover-kindin-2 (green), mRuby-PYCR1 (red), and merged images are shown ( g ). Scale bar = 10 μm. The efficiencies of FRET between the pair of mClover-kindlin-2 and mRuby-PYCR1, or that of mClover-F1 and F3 deletion mutant of kindlin-2 and mRuby-PYCR1 were analyzed ( h , at least n = 15 cells per group were counted from at least three independent experiments). Data are shown as mean ± SEM using two-tailed unpaired Student’s t -test, *** p < 0.001

Article Snippet: Briefly, A549 cell lysates were immunoprecipitated with mouse anti-kindlin-2 antibody (clone 3A3.5 ) or control mouse IgG (Santa Cruz, Cat# sc-2025).

Techniques: Infection, Plasmid Preparation, Sequencing, Western Blot, Expressing, Staining, Confocal Microscopy, Fluorescence, Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Kindlin-2 links mechano-environment to proline synthesis and tumor growth

doi: 10.1038/s41467-019-08772-3

Figure Lengend Snippet: The effects of kindlin-2 depletion on PYCR, P5CS, and PRODH levels. a A549 cells were infected with control (Sh-NC) or kindlin-2 shRNA (Sh-K2) lentivirus for 5 days and then analyzed by western blot with antibodies recognizing kindlin-2, PYCR1, PYCR2, PYCRL, P5CS, PRODH, and GAPDH (as a loading control). Three independent experiments were shown. b Protein levels of kindlin-2, PYCR1, PYCR2, PYCRL, P5CS, and PRODH relative to that of GAPDH in Sh-NC and Sh-K2 cells were quantified by densitometric analysis of western blottings and compared with those in the A549 cells (normalized to 1, n = 3). Data represent mean ± SEM from three independent experiments using one-way ANOVA with Tukey–Kramer post-hoc analyses, *** p < 0.001, NS, no significance

Article Snippet: Briefly, A549 cell lysates were immunoprecipitated with mouse anti-kindlin-2 antibody (clone 3A3.5 ) or control mouse IgG (Santa Cruz, Cat# sc-2025).

Techniques: Infection, Control, shRNA, Western Blot

Journal: Nature Communications

Article Title: Kindlin-2 links mechano-environment to proline synthesis and tumor growth

doi: 10.1038/s41467-019-08772-3

Figure Lengend Snippet: Kindlin-2 regulates PYCR1 and proline levels. a Kindlin-2 KO cells were infected with lentiviral vectors encoding 3xFLAG-tagged kindlin-2 (3fl-K2), 3xFLAG-tagged kindlin-2 F1, and F3 deletion mutant (3fl-K2∆F1∆F3) or 3xFLAG vector lacking kindlin-2 sequence (3fl). Three days later, the cells were analyzed by western blotting. b Protein levels of PYCR1 relative to that of GAPDH were quantified by densitometric analyses. The levels of PYCR1 in KO, 3fl, 3fl-K2, or 3fl-K2∆F1∆F3 cells were compared with that of A549 cells (normalized to 1) ( n = 3). c The cytosolic (lane 3 and 5), mitochondrial (lane 4 and 6), and total (lane 1 and 2) lysates from A549 or kindlin-2 KO cells were analyzed by western blotting. d The cells were immunofluorescently stained with DAPI and anti-PYCR1 antibodies. Scale bar = 10 μm. e The mRNA levels of PYCR1 in kindlin-2 KO cells were analyzed by RT-PCR ( n = 4) and compared with those in A549 cells (normalized to 1). f , g The proline levels were analyzed. The results were visualized ( f ) and quantified ( g , n = 4). Scale bar = 1 cm. h – j A549 cells infected with kindlin-2 shRNA (Sh-K2) or control (Sh-NC) lentivirus were analyzed by western blotting ( h ). The proline levels were analyzed. The results were visualized ( i ) and quantified ( j , n = 3). Scale bar = 1cm. k – m NCI-H358 cells were infected with kindlin-2 shRNA (Sh-K2) or control (Sh-NC) lentivirus for 5 days and then analyzed by western blotting ( k ). l , m The proline levels were analyzed. The results were visualized ( l ) and quantified ( m , n = 3). Scale bar = 1 cm. n , o The effect of kindlin-2 on PYCR1 enzyme activity. The enzyme activity of purified PYCR1 (PY1) with or without GST-kindlin-2 (GST-K2) was analyzed. n The absorbance at 340 nm during the first 5 min of the reaction. o The relative activities of PYCR1 with or without GST-Kindlin-2 were calculated from n ( n = 3). Data in b , e , g , j , m , and o represent means ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis in b , j , and m or using two-tailed unpaired Student’s t -test in e , j , and o , ** P < 0.01; *** p < 0.001. NS, no significance

Article Snippet: Briefly, A549 cell lysates were immunoprecipitated with mouse anti-kindlin-2 antibody (clone 3A3.5 ) or control mouse IgG (Santa Cruz, Cat# sc-2025).

Techniques: Infection, Mutagenesis, Plasmid Preparation, Sequencing, Western Blot, Staining, Reverse Transcription Polymerase Chain Reaction, shRNA, Control, Activity Assay, Purification, Two Tailed Test

Journal: Nature Communications

Article Title: Kindlin-2 links mechano-environment to proline synthesis and tumor growth

doi: 10.1038/s41467-019-08772-3

Figure Lengend Snippet: Depletion of kindlin-2 increases ROS production and apoptosis. a – c A549 cells were infected with K2 shRNA (Sh-K2) lentivirus or control lentivirus (Sh-NC). Two days after the infection, the cells were infected with lentiviral expression vectors encoding 3xFLAG-tagged kindlin-2 (3fl-K2) or 3xFLAG empty vector (3fl). Three days after the lentiviral infection, the cells were analyzed by western blotting with antibodies recognizing K2 or GAPDH (as a loading control) ( a ). The levels of ROS were analyzed using DHE fluorescence probe as described in the Methods ( b ). Scale bar, 75 μm. The mean fluorescence intensities (MFI) were calculated using the Image-pro plus software from two different experiments ( c , at least n = 15 field per group were counted from at least three independent experiments). d The cells (as specified in the figure) were immunofluorescently stained with DAPI and anti-cleaved caspase-3 antibody. Scale bar = 25 μm. The percentages of cleaved caspase-3-positive cells were quantified ( e ) as described in the Methods. Replicates ( n = 5) from one representative experiment are shown. Data in c and e are presented as mean ± SEM using one-way ANOVA with Tukey–Kramer post-hoc analysis, * P < 0.05; *** p < 0.001

Article Snippet: Briefly, A549 cell lysates were immunoprecipitated with mouse anti-kindlin-2 antibody (clone 3A3.5 ) or control mouse IgG (Santa Cruz, Cat# sc-2025).

Techniques: Infection, shRNA, Control, Expressing, Plasmid Preparation, Western Blot, Fluorescence, Software, Staining

Journal: Nature Communications

Article Title: Kindlin-2 links mechano-environment to proline synthesis and tumor growth

doi: 10.1038/s41467-019-08772-3

Figure Lengend Snippet: Kindlin-2 regulates cell proliferation through PYCR1. a – d Kindlin-2 KO A549 cells were infected with lentiviral vectors encoding 3xFLAG-tagged kindlin-2 (3fl-K2) or 3xFLAG empty vector (3fl). Three days later, the cells were analyzed by western blotting ( a ). The cells were seeded in six-well plate (1 × 10 5 /well), cultured for 2 days, and the number of cells was counted ( b , n = 3). Cells were stained with DAPI and anti-Ki67 antibody ( d ). Scale bar, 75 μm. The percentages of Ki67-positive cells were quantified ( c , n = 5). e , f Kindlin-2 KO A549 cells were treated with proline for 3 days (interval of treatment = 12 h). Cells were stained with DAPI and anti-Ki67 antibody ( e ). Scale bar, 75 μm. The percentages of Ki67-positive cells were quantified ( f , n = 5). g, h A549 cells were infected with PYCR1 shRNA (Sh-PY1) or control (Sh-NC) lentivirus for 5 days. The cells were seeded in six-well plate (1×10 5 /well), cultured for 2 days, and the numbers of cells were counted ( g , n = 3). Cells were stained with DAPI and anti-Ki67 antibody and the percentages of Ki67-positive cells were quantified ( h , n = 5). i – l Kindlin-2 KO A549 cell lines (ko) were infected with lentiviral vectors encoding 3×FLAG tagged PYCR1 (3fl-PY1) or 3xFLAG empty vector (3fl). Three days later, the cells were analyzed by western blotting ( i ). The proline levels in KO, KO+3fl, and KO+3fl-PY1 cells were quantified and compared with that of A549 ( j , n = 3). The cells were seeded in growth medium in six-well plate (1×10 5 /well), cultured for 2 days, and then the numbers of cells were counted ( k , n = 3). Cells were stained with DAPI and anti-Ki67 antibody. The percentages of Ki67-positive cells were quantified ( l , n = 5). m – o Kindlin-2 KO A549 cells were infected with lentiviral vectors encoding 3xFLAG-tagged PYCR1 (3fl-PY1) or 3xFLAG vector (3fl). Three days later, the cells were analyzed by western blotting ( m ). Cells were immunofluorescently stained with Alexa Fluor-conjugated phalloidin ( n ). Scale bar, 25 μm. Cell areas ( o ) were quantified using Image J. At least 50 cells from each group were analyzed ( n = 5). Data in b , c , f , g , h , j – l , and o are presented as mean ± SEM using one-way ANOVA with Tukey–Kramer post-hoc analysis, * P < 0.05; ** P < 0.01; *** p < 0.001

Article Snippet: Briefly, A549 cell lysates were immunoprecipitated with mouse anti-kindlin-2 antibody (clone 3A3.5 ) or control mouse IgG (Santa Cruz, Cat# sc-2025).

Techniques: Infection, Plasmid Preparation, Western Blot, Cell Culture, Staining, shRNA, Control

Journal: Nature Communications

Article Title: Kindlin-2 links mechano-environment to proline synthesis and tumor growth

doi: 10.1038/s41467-019-08772-3

Figure Lengend Snippet: ECM stiffening regulates kindlin-2-PYCR1 interaction. a The cytosolic (lanes 3 and 5), mitochondrial (lanes 4 and 6), and total (lanes 1 and 2) lysates from A549 cells on stiff (elastic modulus of 40 kPa) or soft (elastic modulus of 0.35 kPa) hydrogels were analyzed by western blotting. b A549 cells plated on stiff or soft hydrogels for 48 h were analyzed by IP and western blotting. Lane 3, the sample was prepared as that of lane 4, except anti-kindlin-2 antibody was substituted with irrelevant mouse IgG. c A549 cells expressing mClover-kindin-2 and mRuby-PYCR1 on stiff or soft hydrogels were analyzed by FLIM-FRET (> 15 cells per group were counted from at least three independent experiments). d , e A549 cells on soft hydrogels were pretreated with or without 10 mM MBCD for 1 h and stained with anti-β1 integrin antibody ( d ) or analyzed by IP with anti-kindlin-2 antibody ( e ). Scale bar in d , 25 μm. The lysates (lanes 1 and 2), control IgG (lane 3), and anti-kindlin-2 immunoprecipitates (lanes 4 and 5) were analyzed by western blotting ( e ). f – l A549 or kindlin-2 KO cells on stiff or soft hydrogels were analyzed by western blotting ( f ). Protein levels of PYCR1 relative to that of GAPDH were quantified by densitometric analyses and compared with that of A549 cells (normalized to 1) ( g , n = 3). The mRNA level of PYCR1 in A549 cells on soft hydrogels was analyzed by RT-PCR and compared with that in A549 cells on stiff hydrogels (normalized to 1) ( h , n = 3). The proline levels in A549 or kindlin-2 KO cells on stiff or soft hydrogels were analyzed ( i , n = 3). The cells (3.5 × 10 4 /gel) were plated on stiff or soft hydrogels, cultured for 2 days, the number was counted ( j , n = 3) and cells were stained with DAPI and anti-Ki67 antibody ( k ). Scale bar, 75 μm. The percentages of Ki67-positive cells were quantified ( l , n = 5). Data in c , g , h , i , j , and l represent means ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis ( g , i , j , l ) or two-tailed unpaired Student’s t -test ( c , h ). *** p < 0.001. NS, no significance

Article Snippet: Briefly, A549 cell lysates were immunoprecipitated with mouse anti-kindlin-2 antibody (clone 3A3.5 ) or control mouse IgG (Santa Cruz, Cat# sc-2025).

Techniques: Western Blot, Expressing, Staining, Control, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Two Tailed Test

Journal: The Journal of experimental medicine

Article Title: Diphtheria toxin activates ribotoxic stress and NLRP1 inflammasome-driven pyroptosis.

doi: 10.1084/jem.20230105

Figure Lengend Snippet: Figure 2. DT and C. diphtheriae trigger RSR downstream of EEF2 inactivation. (A) Immunoblot of ZAKα, p38 NLRP1 in primary keratinocytes treated with the indicated triggers in the presence of DMSO or M443 (1 µM). M443 was added 10 min before DT or bacterial filtrate. Lysates were harvested 3 h after treatment. Immunoblot representative of three replicate experiments. (B) Immunoblot of RSR kinases and GFP-NLRP1linker in control and ZAKα KO N/ TERT cells. ANS-treated cells were harvested 2 h after treatment. Indicated cells were primed with 25 ng/ml TNFα overnight and then treated with 150 ng/ml DT. Immunoblot representative of three replicate experiments. Source data are available for this figure: SourceData F2.

Article Snippet: 293Ts (#CRL-3216; ATCC) and A549-ASC-GFP-NLRP1 (a549ascg-nlrp1; Invivogen) were cultured according to manufacturer’s protocols.

Techniques: Western Blot, Control

Journal: The Journal of experimental medicine

Article Title: Diphtheria toxin activates ribotoxic stress and NLRP1 inflammasome-driven pyroptosis.

doi: 10.1084/jem.20230105

Figure Lengend Snippet: Figure 3. DT and C. diphtheriae cause pyroptosis in a ZAKα and NLRP1-dependent manner but also cause other forms of cell death. (A) Comparison of PI uptake kinetics between control and NLRP1 KO primary keratinocytes in response to purified DT. Error bars represent three biological replicates, with each drug treatment considered as one replicate. Significance values were calculated from Student’s t test at the 7-h time point. **, P ≤0.01. (B) Immunoblot of GSDMD, cleaved caspase-3, and IL-1β in WT and NLRP1 KO keratinocyte lysates or media 24 h after the indicated treatment. Immunoblot is representative of three replicate experiments. (C) Comparison of PI uptake kinetics between control and ZAKα KO primary keratinocytes in response to purified DT. Error bars represent three biological replicates, with each drug treatment considered as one replicate. Significance values were calculated from Student’s t test at the 7-h time point. *, P ≤0.05. (D) Immunoblot of ZAKα, GSDMD, MCL-1, cleaved caspase-3 and IL-1β in WT and ZAKα KO keratinocyte lysates or media 24 h after the indicated treatment. Salinomycin (10 µM) does not activate the NLRP1 inflammasome and was used as a negative control. Immunoblot is representative of three replicate experiments. Source data are available for this figure: SourceData F3.

Article Snippet: 293Ts (#CRL-3216; ATCC) and A549-ASC-GFP-NLRP1 (a549ascg-nlrp1; Invivogen) were cultured according to manufacturer’s protocols.

Techniques: Comparison, Control, Purification, Western Blot, Negative Control