Journal: Heliyon

Article Title: Augmented antitumor immune responses of HER2-targeted pyroptotic induction by long-lasting recombinant immunopyroptotins

doi: 10.1016/j.heliyon.2024.e30444

Figure Lengend Snippet: Schematic illustration of the use of immunopyroptotins for antitumor therapy by HER2-targeted induction of pyroptosis. The immunopyroptotins consist of the humanized anti-HER2 single-chain antibody P1h3, albumin-binding peptide (ABD035 or dAb7h8), cathepsin B-cleavable peptide B2, endosome-disruptive peptide E5C3, and active pyroptotic effector gasdermin D-N fragment (GSDMD-N). The fusion proteins are expressed in 293F cells and purified using Ni 2+ -NTA affinity chromatography. The immunopyroptotins are then injected into a unilateral tumor in immunocompetent mice with bilateral xenograft breast tumors. Upon recognition and subsequent endocytosis into HER2-positive tumor cells, the immunopyroptotins are cleaved at the B2 site by cathepsin B in the endosomes, and GSDMD-N is released into the cytosol via E5C3-mediated endosomal escape, finally inducing tumor cell pyroptosis. Systemic immune responses are elicited by the accumulation of CD8 + T cells, mature DCs, and upregulated expression of proinflammatory cytokines, leading to sustained remission of the distant tumor as well as the injected tumor.

Article Snippet: PC9 lung adenocarcinoma cell lines (CVCL_B260; catalogue number: 305045; human lung adenocarcinoma cells without HER2 expression) were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany); of them, 293F cells were cultured in FreeStyle 293F expression medium (Gibco, Waltham, MA, USA), and SKBR-3 cells were grown in McCoy's 5A medium supplemented with 10 % (v/v) fetal bovine serum (FBS) (Sigma-Aldrich, Merck KGaA, Germany) and 1 % (v/v) penicillin–streptomycin (Hyclone, Logan, UT).

Techniques: Binding Assay, Purification, Affinity Chromatography, Injection, Expressing

Journal: Heliyon

Article Title: Augmented antitumor immune responses of HER2-targeted pyroptotic induction by long-lasting recombinant immunopyroptotins

doi: 10.1016/j.heliyon.2024.e30444

Figure Lengend Snippet: Killing capacity of the long-lasting immunopyroptotins in vitro. (A) Viability of SKBR-3, N87, NCI–H1975, and PC9 cells incubated with 1 mg/mL purified proteins for 12 and 24 h. (B, C) LDH release (B) and Annexin V/PI flow cytometry analyses of (C) of SKBR-3 cells incubated with 1 mg/mL purified proteins for 24 h. (D) Pyroptotic morphology of SKBR-3 cells treated with the indicated immunopyroptotins for 12 h. Data are expressed as mean ± standard error of the mean. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by t -test.

Article Snippet: PC9 lung adenocarcinoma cell lines (CVCL_B260; catalogue number: 305045; human lung adenocarcinoma cells without HER2 expression) were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany); of them, 293F cells were cultured in FreeStyle 293F expression medium (Gibco, Waltham, MA, USA), and SKBR-3 cells were grown in McCoy's 5A medium supplemented with 10 % (v/v) fetal bovine serum (FBS) (Sigma-Aldrich, Merck KGaA, Germany) and 1 % (v/v) penicillin–streptomycin (Hyclone, Logan, UT).

Techniques: In Vitro, Incubation, Purification, Flow Cytometry

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: IC 50 values (nM) for the mutant EGFR-expressing Ba/F3 cells, PC9 cells or MGH121 cells.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Mutagenesis, Expressing

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a ) The results of screening the growth-inhibitory activity of 30 drugs in Ba/F3 cells expressing four types of EGFR-del19 with or without T790M or C797S mutations are shown in a heat map. Ba/F3 cells expressing each EGFR mutant were treated with 100 nM of the indicated inhibitors. After 72 h of drug treatment, the cell viability was measured using the CellTiter-Glo assay. Relative cell viability was calculated from each value divided by the DMSO control. Among the inhibitors, only brigatinib and ponatinib were sufficiently efficacious against the triple-mutant EGFR. AZD3463 acted as a weak inhibitor to the triple mutation. ( b ) Growth inhibition assessed by the CellTiter-Glo assay of EGFR-C797S/T790M/del19 (triple-del19)-mutated Ba/F3 cells treated with gefitinib, osimertinib and brigatinib.; N =3. Results are expressed as mean±s.d. IC 50 values were calculated using growth inhibition assay. ( c ) Phosphorylation of EGFR and downstream signals were significantly inhibited by brigatinib in Ba/F3 cells expressing triple-del19 even though afatinib and osimertinib did not suppress at all the EGFR signalling of triple-del19.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Activity Assay, Expressing, Mutagenesis, Glo Assay, Control, Inhibition, Growth Inhibition Assay, Phospho-proteomics

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a ) Chemical structures of six ALK–TKIs were very similar. ( b , c ) IC 50 values in Ba/F3 cells expressing four mutation types of EGFR-del19 were obtained by treatment with brigatinib, AP26113-analog, AZD3463, TAE684, ceritinib and ASP3026 for 72 h. Those of C797S/T790M/del19 were shown by bar graph ( b ) and those of all mutation types were demonstrated by a table ( c ). The CellTiter-Glo assay was used to measure cell viability. ( d , e ) Ba/F3 cells expressing T790M/del19 ( d ) or C797S/T790M/del19 ( e ) were treated with the indicated concentrations of brigatinib, AP26113 analog, TAE684, ceritinib or ASP3026 for 6 h. Phosphorylation of EGFR and its downstream signals were evaluated by western blotting with the indicated antibodies.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Expressing, Mutagenesis, Glo Assay, Phospho-proteomics, Western Blot

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a – e ) PC9 (del19) ( a ), PC9-T790M (T790M/del19) ( b ), PC9-triple mutant (C797S/T790M/del19) ( c ), MGH121 parent (T790M/del19) ( d ) and MGH121 resistant-2 (C797S/T790M/del19) ( e ) cells were treated with serially diluted gefitinib, osimertinib and brigatinib for 72 h. Cell viability was measured using the CellTiter-Glo assay.; N =3. Results are expressed as mean±s.d. ( f ) Western blotting of PC9 triple mutant (C797S/T790M/del19) cells indicated that brigatinib and AP26113 analog, but not afatinib or osimertinib, suppressed phosphorylation of EGFR and its downstream signalling. ( g ) Similar results were obtained in MGH121 resistant-2.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Mutagenesis, Glo Assay, Western Blot, Phospho-proteomics

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a ) The cell growth inhibition of Ba/F3 cells expressing EGFR-C797S/T790M/del19 (EGFR-triple-del19) treated with brigatinib, AP26113-analog, AZD3463 and osimertinib at indicated concentrations combined with or without cetuximab (10 μg ml −1 ) for 72 h assessed by CellTiter-Glo assay. ( b ) Inhibition of EGFR signal pathway in BaF3 EGFR-triple-del19 cells treated with brigatinib+cetuximab (10 μg ml −1 ) for 6 h was evaluated using western blotting. ( c , d ) The cell growth inhibition of PC9 triple-mutant cells ( c ) and MGH121-res2 cells ( d ) treated with brigatinib and osimertinib at indicated concentrations combined with or without cetuximab (10 μg ml −1 ) for 72 h assessed by CellTiter-Glo assay. ( e , f ) Inhibition of EGFR signal pathway in PC9 triple-mutant cells ( e ) and MGH121-res2 cells ( f ) treated with brigatinib+cetuximab (10 μg ml −1 ) for 6 h was evaluated using western blotting.; Results in a , c , e are expressed as mean±s.d. ( N =3). The significance of difference between indicated groups are calculated by Student's t -test (NS; not significant, * P <0.05, ** P <0.01).

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Inhibition, Expressing, Glo Assay, Western Blot, Mutagenesis

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a , b ) PC9 cells expressing EGFR-C797S/T790M/del19 were subcutaneously implanted into Balb-c nu/nu mice. When the average tumour volume reached ∼200 mm 3 , the mice were randomized into vehicle control or treatment groups (50 mg kg −1 of osimertinib, 75 mg kg −1 of brigatinib, 1 mg per mouse of cetuximab three times a week or 75 mg kg −1 of brigatinib combined with cetuximab administered as previously described) and treated once daily by oral gavage for the indicated period. Tumour volume ( V ) was calculated as 0.5 × length × width 2 , and body weights (B.W.) of mice were measured twice weekly.; N =6. Results are expressed as mean±s.d. The significance of difference between the mean tumour volume of control and of brigatinib on day 7, between brigatinib and brigatinib+cetuximab on day 23, respectively, are calculated by Mann–Whitney U test (** P <0.01). ( c ) Survival periods of mice in each treatment arm were demonstrated using the Kaplan–Meier curve. ( d ) Phosphorylation of EGFR and its downstream signalling in two tumour samples obtained from each group were evaluated using western blotting. ( e , f ) In vivo experiment of PC9 triple-mutant cells following a similar protocol as in , using panitumumab 0.5 mg per mouse two times a week administered peritoneally instead of cetuximab.; N =6. Results are expressed as mean±s.d. The significance of difference between the mean tumour volume of control and of brigatinib on day 16, between brigatinib and brigatinib+panitumumab on day 23, respectively, are calculated by Mann–Whitney U test (** P <0.01). ( g ) A Kaplan–Meier curve of the survival of the mice in each treatment arm. ( h ) Phosphorylation of EGFR and its downstream signalling in two tumour samples obtained from xenografts of PC9-triple mutant cells treated for 8 days with the indicated drugs (brigatinib: 75 mg kg −1 daily, administered orally; panitumumab: 0.5 mg per mouse two times a week, administered peritoneally) were assessed by western blotting with the indicated antibodies.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Expressing, Control, MANN-WHITNEY, Phospho-proteomics, Western Blot, In Vivo, Mutagenesis

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a , b ) MGH121-res2 expressing EGFR-C797S/T790M/del19 were subcutaneously implanted into SCID-beige mice. When the average tumour volume reached ∼200 mm 3 , the mice were randomized into vehicle control and treatment groups (50 mg kg −1 of osimertinib (po), 75 mg kg −1 of brigatinib (po), 1 mg per mouse of cetuximab two times a week and 75 mg kg −1 of brigatinib combined with cetuximab administered as previously described, respectively) and treated for the indicated period. Tumour volume ( V ) was calculated as 0.5 × length × width 2 , and the body weights (B.W.) of the mice were measured twice weekly. N =6. Results are expressed as mean±s.d. The significance in difference between the mean tumour volume of control and of osimertinib, brigatinib and cetuximab, between cetuximab and brigatinib+cetuximab, respectively, on day 42 are calculated by Mann–Whitney U test (NS: not significant, * P <0.05, ** P <0.01).

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Expressing, Control, MANN-WHITNEY

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The knockdown of FGL1 and RNA-seq results. (A) The knockdown of FGL1 confirmed by western blot. (B) The knockdown of FGL1 confirmed by immunofluorescent staining. (C) Volcano map of differential gene changes in PC9 cells after FGL1 knockdown. (D) Volcano map of differential gene changes in Jurkat T cells after FGL1 knockdown. (E) The enrichment of differential genes in PC9 cells, analyzed by GSEA. **, P<0.01; ***, P<0.001. NC, negative control; KD, knockdown; LUAD, lung adenocarcinoma; FGL1, fibrinogen-like protein 1.

Article Snippet: PC9 lung cancer cells and HCC827 lung cancer cells (human; iCell Bioscience Inc., Shanghai China) were cultured in RPMI 1640 (Gibco, Shanghai, China) with 10% fetal calf serum at 37 °C in 5% CO 2 .

Techniques: Knockdown, RNA Sequencing, Western Blot, Staining, Negative Control

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The experiments related to cell proliferation. (A,B) Effects of FGL1 knockdown on the cycle of PC9 cells and Jurkat T cells. (C) Real-time cell analyzer presents the real-time condition of cell proliferation. (D) Colony formation confirms the cell proliferation of PC9 cells and HCC827 cells, the magnifications in the figure are 40× and 200×, respectively, both of them were stained with crystal violet. **, P<0.01; ***, P<0.001; ****, P<0.0001. NC, negative control; KD, knockdown; LUAD, lung adenocarcinoma; FGL1, fibrinogen-like protein 1.

Article Snippet: PC9 lung cancer cells and HCC827 lung cancer cells (human; iCell Bioscience Inc., Shanghai China) were cultured in RPMI 1640 (Gibco, Shanghai, China) with 10% fetal calf serum at 37 °C in 5% CO 2 .

Techniques: Knockdown, Staining, Negative Control

Journal: Cancer Science

Article Title: EGFR inhibition in EGFR ‐mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment

doi: 10.1111/cas.15701

Figure Lengend Snippet: CD24 expression is induced in EGFR‐mutant NSCLC cells upon EGFR‐TKI treatment. (A) Differential gene expression between EGFR‐TKI‐treated versus ‐untreated EGFR‐mutant NSCLC cells in GSE75308 (left) and GSE57156 (right) were analyzed. (B) EGFR‐mutant PC9 and H1975 cells, and EGFR‐wild‐type RERF‐LC‐Ad1 and H522 cells were treated with osimertinib for 72 h in vitro. CD24 expression was analyzed by flow cytometry. Gray: isotype control; dotted line: untreated; line: treated. Data are representative of four independent experiments. (C) Numbers of CD24 molecules expressed on cells with or without EGFR‐TKI treatment in vitro. Data are presented as mean ± SEM from four independent experiments. Numbers of the receptors were calculated using the BD QuantiBrite kit as described in Materials and Methods. (D) CD24 gene expression in tumor cells with or without EGFR‐TKI treatment in vitro were analyzed by qRT‐PCR. Data are presented as mean ± SEM of technical replicates from at least two independent experiments. (E) EGFR‐mutant PC9 and EGFR‐wild‐type H522 cells were treated either with osimertinib, gefitinib, or afatinib for 72 h in vitro. CD24 expression was analyzed by flow cytometry. Data are representative of two independent experiments. * q < 0.01.

Article Snippet: The lung cancer cell line, PC9, was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Expressing, Mutagenesis, Gene Expression, In Vitro, Flow Cytometry, Control, Quantitative RT-PCR

Journal: Cancer Science

Article Title: EGFR inhibition in EGFR ‐mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment

doi: 10.1111/cas.15701

Figure Lengend Snippet: CD24 induction in EGFR‐mutant NSCLC cells is a target of antibody‐dependent cellular phagocytosis. (A) The fraction of immune cell types in the tumor microenvironment of NSCLC from the TCGA‐LUAD dataset (B) Arbitrary gene expression levels of SIGLEC‐10 and MRC1 across macrophages in the tumor microenvironment of NSCLC (C) Siglec‐10 expression was analyzed by flow cytometry. Gray: isotype control, line: Siglec‐10. Human monocyte‐derived macrophages were differentiated according to the protocols shown in Supplementary Table . Data are a representative of at least two independent experiments from three healthy donors. (D) M‐CSF differentiated macrophages and EGFR‐TKI‐treated PC9 cells were incubated for 9 h with anti‐CD24 antibodies or isotype control antibodies. Cytochalasin D was used to inhibit macrophage phagocytosis as described in Materials and Methods. Data are presented as the mean ± SEM of technical replicates from at least two independent experiments. *** p < 0.001.

Article Snippet: The lung cancer cell line, PC9, was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Mutagenesis, Gene Expression, Expressing, Flow Cytometry, Control, Derivative Assay, Incubation

Journal: Cancer Science

Article Title: EGFR inhibition in EGFR ‐mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment

doi: 10.1111/cas.15701

Figure Lengend Snippet: EGFR‐TKIs accelerate the release of the cell‐free DNA activating type I IFN response in a STING‐dependent manner. (A) EGFR‐mutant tumor cells were treated with either osimertinib (EGFR‐TKI) or paclitaxel. Data are presented as the mean ± SEM of technical triplicates from at least two independent experiments. (B) cfDNA concentrations in the supernatant were measured as described in Materials and Methods. Data are presented as the mean ± SEM of technical triplicates from at least two independent experiments. (C) THP‐1‐Dual and THP‐1‐Dual STING‐knockout (KO) cells were cultured either with LPS (500 ng/ml), IFNa2a (10,000 U/ml), tumor‐derived cfDNA (PC9 and H1975 cfDNA)/lipofectamine, or lipofectamine alone. IRF (left) and NF‐κB (right) activities were analyzed as described in Materials and Methods. (D) PMA‐differentiated THP‐1 monocytes were pretreated either with lipofectamine, poly(dA:dT) (100 ng/ml), tumor‐derived cfDNA (PC9) (100 ng/ml)/lipofectamine, or IFNa2a (20,000 U/ml) followed by IFN‐γ stimulation (100 ng/ml). (E) Expression levels of CXCL9 and CXCL10 were analyzed using flow cytometry. * p < 0.05, ** p < 0.01, **** p < 0.0001, * q < 0.01. ns, not significant.

Article Snippet: The lung cancer cell line, PC9, was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Mutagenesis, Knock-Out, Cell Culture, Derivative Assay, Expressing, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Rho family small GTPase Rif regulates Wnt5a-Ror1-Dvl2 signaling and promotes lung adenocarcinoma progression

doi: 10.1016/j.jbc.2023.105248

Figure Lengend Snippet: Ror1 and Rif are colocalized at filopodia in LUAD cells. A, Western blot analysis of Ror1, Ror2, and Rif in LUAD cell lines, showing expression of both Ror1 and Rif were detectable in all LUAD cell lines examined. Blots are representative of two independent experiments. B, coimmunoprecipitation assay, showing association between Rif and Ror1 at endogenous protein levels in the indicated LUAD cells. Whole-cell lysates were subjected to immunoprecipitation (IP) with anti-Ror1 antibody or control immunoglobulin G (isotype matched), followed by Western blotting. Blots are representative of six (PC9), three (HCC827), and two (A549) independent experiments. C and D, representative xz-images of PC9 cells expressing YFP-Rif and Ror1-mCh on a 2D surface ( C ) or Matrigel ( D ), showing colocalization of these proteins at filopodia. Phalloidin staining in ( D ) shows highly accumulated F-actin on the front side containing filopodia. In ( D ), the xy images ( lower panels ) sectioned along the white arrow in xz confocal image ( upper panels ) are shown. Images are representative of at least three independent experiments. The scale bars represent 10 μm. Magnified images of boxed regions are shown on the right . The scale bars in magnified images represent 2 μm ( C ) and 5 μm ( D ). LUAD, lung adenocarcinoma; Rif, Rho in filopodia.

Article Snippet: PC9 cells (Control, Ror1 KO #1/#2, Rif KO #1/#2) at 2.5 × 10 5 in 50% (v/v) Matrigel in PBS were subcutaneously transplanted into 6-week-old nude mice (BALB/cSlc- nu/nu ) obtained from Japan SLC.

Techniques: Western Blot, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: Rho family small GTPase Rif regulates Wnt5a-Ror1-Dvl2 signaling and promotes lung adenocarcinoma progression

doi: 10.1016/j.jbc.2023.105248

Figure Lengend Snippet: Ror1 and Rif promote cell proliferation, survival, and invasion in vitro and tumor development in vivo . A, Western blot analysis showing efficient knockdown of Ror1 and Rif by the respective siRNAs in PC9 cells. Images are representative of at least three independent experiments. B and C, effects of knockdown of Ror1 or Rif on cell proliferation ( B ) and survival ( C ). Viability of PC9 cells transfected with the indicated siRNAs were assessed in media containing 10% ( B ) or 1% ( C ) FBS by using the WST-8 assay, as described in Materials and methods. In ( C ), cells were cultured on either a PLL-coated 2D surface or Matrigel for 3 days before measuring cell viability. Data are expressed as mean ± SD of three independent experiments, each performed in triplicate ( B ) or mean ± SD of three technical replicates of a representative experiment out of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, Tukey’s test. D, Transwell invasion assay showing decreased invasion of PC9 cells treated with si- Ror1 or si- Rif . Data are expressed as mean ± SD of three independent experiments. ∗∗∗ p < 0.001, Dunnett’s test. E, Western blot analysis showing ablated expression of Ror1 and Rif in the respective KO PC9 cells. Blots are representative of two independent experiments, respectively. F, representative images of tumor tissues generated by subcutaneous transplantation of control, Ror1 -KO, or Rif -KO PC9 cells into nude mice. Graph shows mean weight of tumor tissues. Data are expressed as mean ± SD of seven (control) and three ( Ror1 KO#1, #2, and Rif KO#1, #2) xenografts from three and seven mice, respectively. ∗∗ p < 0.01, ∗∗∗ p < 0.001, Dunnett’s test. PLL, poly-L-lysine; Rif, Rho in filopodia.

Article Snippet: PC9 cells (Control, Ror1 KO #1/#2, Rif KO #1/#2) at 2.5 × 10 5 in 50% (v/v) Matrigel in PBS were subcutaneously transplanted into 6-week-old nude mice (BALB/cSlc- nu/nu ) obtained from Japan SLC.

Techniques: In Vitro, In Vivo, Western Blot, Knockdown, Transfection, Cell Culture, Transwell Invasion Assay, Expressing, Generated, Transplantation Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Rho family small GTPase Rif regulates Wnt5a-Ror1-Dvl2 signaling and promotes lung adenocarcinoma progression

doi: 10.1016/j.jbc.2023.105248

Figure Lengend Snippet: Ror1 and Rif are required to establish front–rear polarity and invasive activity on Matrigel. A and B, PC9 cells were cultured on Matrigel for 1 h. Representative xz-images of the cells showing protrusion of filopodia ( yellow arrowheads ) in control cells ( A ). Yellow dotted lines indicate the surface of the Matrigel. Images are representative of three independent experiments. Number of filopodia are quantified ( B ). Data are presented as a box-and-whisker plot . n = 31 (si-Ctrl, si- Ror1 #1), 38 (si- Ror1 #2), 24 (si- Rif #1), and 21 (si- Rif #2) cells from three independent experiments. ∗∗∗ p < 0.001, Dunnett’s test. C and D, filopodia formation induced by ectopic expression of Rif(QL) and Ror1 was suppressed by siRNAs against Ror1 and Rif, respectively. PC9 cells stably expressing Flag-Rif(QL) ( C ) or Ror1-mCherry ( D ), respectively, were transfected with siRNAs against Ror1 or Rif as indicated and assessed for filopodia formation as shown in ( A ). Data are presented as a box-and-whisker plot . n = 28 to 38 ( C ) and 23 to 34 cells ( D ) from three independent experiments, respectively. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, Tukey’s test. E and F, PC9 cells were cultured on Matrigel containing DQ-collagen IV for 2 h. Representative 3D images ( upper panels ) and xy images sectioned around the bottom surface of the cells ( lower panels ) are shown. Yellow arrowheads indicate filopodia in control cells. Images are representative of three independent experiments. Intensity of the degraded DQ-collagen IV was quantified ( F ). Data are presented as a box-and-whisker plot . n = 57 (si-Ctrl), 60 (si- Ror1 #1), 55 (si- Ror1 #2), 59 (si- Rif #1), 68 (si- Rif #2) cells from three independent experiments. ∗∗∗ p < 0.001, Dunnett’s test. G and H, PC9 cells were cultured on Matrigel for 30 min, and the Golgi apparatus (GM130) and nucleus (4′,6-diamidino-2-phenylindole) were stained. Representative xz-image stacked along the y -direction are shown ( G , upper panel ). Images are representative of three independent experiments. Schematic ( G , lower panel ) shows the front and rear of a cell ( green ) defied as the 120° sectors emerging from the center of the nucleus toward the front and rear. Fluorescence intensity of GM130 within the front and rear sectors were quantified ( H ). Dotted lines in the graph indicate the level of expected random orientation of 33%. Data are presented as a box-and-whisker plot . n = 178 (si-Ctrl, si- Ror1 #2) and 177 (si- Ror1 #1, si- Rif #1, si- Rif #2) cells from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, Dunnett’s test. The scale bars represent 10 μm ( A and E ) and 5 μm ( G ). DQ, dye-quenched; Rif, Rho in filopodia.

Article Snippet: PC9 cells (Control, Ror1 KO #1/#2, Rif KO #1/#2) at 2.5 × 10 5 in 50% (v/v) Matrigel in PBS were subcutaneously transplanted into 6-week-old nude mice (BALB/cSlc- nu/nu ) obtained from Japan SLC.

Techniques: Activity Assay, Cell Culture, Control, Whisker Assay, Expressing, Stable Transfection, Transfection, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Rho family small GTPase Rif regulates Wnt5a-Ror1-Dvl2 signaling and promotes lung adenocarcinoma progression

doi: 10.1016/j.jbc.2023.105248

Figure Lengend Snippet: Rif is required to activate Wnt5a-Ror1-Dvl2 signaling in PC9 cells cultured on Matrigel. A–D, untreated PC9 cells ( A and B ) or PC9 cells treated with the indicated siRNAs ( C and D ) were cultured on either a PLL-coated plate ( A and B ) or Matrigel ( A – D ). Cells were lysed and analyzed by Western blotting. For dephosphorylation of Dvl2 and Ror1, cell lysates were treated with CIP before subjected to Western blotting ( B ). Black and white arrowheads in anti-Ror1 blots represent the 130 and 115 kDa forms of Ror1, respectively. Blots are representative of three independent experiments. Ratios of phosphorylated and shifted Dvl2 (PS-Dvl2) to unshifted Dvl2 (Dvl2) are shown as mean ± SD of three independent experiments. ∗∗ p < 0.01, ∗∗∗ p < 0.001, t test ( A ), Dunnett’s test ( C and D ). E, PC9 cells transfected with Rif siRNAs were cultured in cell-culture plates and subjected to cell surface biotinylation. Biotin-labeled surface proteins isolated by streptavidin pulldown and whole-cell lysates (WCL) were analyzed by Western blotting. Blots are representative of three independent experiments. CIP, calf intestinal alkaline phosphatase; Dvl, Dishevelled; PLL, poly-L-lysine; PS-Dvl, phosphorylated and shifted Dvl2; Rif, Rho in filopodia.

Article Snippet: PC9 cells (Control, Ror1 KO #1/#2, Rif KO #1/#2) at 2.5 × 10 5 in 50% (v/v) Matrigel in PBS were subcutaneously transplanted into 6-week-old nude mice (BALB/cSlc- nu/nu ) obtained from Japan SLC.

Techniques: Cell Culture, Western Blot, De-Phosphorylation Assay, Transfection, Labeling, Isolation

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet: Genome editing outcomes in individual PC9 cells (A) Genome editing outcomes in isolated clones derived from single PC9 cells edited by HypaCas9 and the single-stranded donor DNA targeting RBM20. Each bar represents one clone, and the proportions of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) in one clone are also shown in each bar. (B) Comparison of the proportions of WT and full NHEJ clones between the mathematical models with three copies of RBM20 and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate the difference. ∗p <0.05. NS: not significantly different (p >0.1). (C) Genome editing outcomes in isolated clones derived from single PC9 cells edited by HypaCas9 and the single-stranded donor DNA targeting ATP7B. Each bar represents one clone, and the proportions of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) in one clone are also shown in each bar. (D) Comparison of the proportions of WT and full NHEJ clones between the mathematical models with three copies of ATP7B and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. ∗p <0.05. NS: not significantly different (p >0.1). (E) Genome editing outcomes in isolated clones derived from single PC9 cells edited by HypaCas9 and the single-stranded donor DNA targeting GRN. Each bar represents one clone, and the proportions of WT (green), NHEJ (blue), HDR (red), and HDR + NHEJ (purple) in one clone are also shown in each bar. (F) Comparison of the proportions of WT and full NHEJ clones between the mathematical models with three copies of GRN and the actually observed cells. Values ±S.E. are shown (n = 3). Student’s t test was used to evaluate differences. NS: not significantly different (p >0.1).

Article Snippet: PC9 cells , RIKEN BioResource Research Center , RCB4455.

Techniques: Isolation, Clone Assay, Derivative Assay, Comparison

Journal: iScience

Article Title: Genome editing is induced in a binary manner in single human cells

doi: 10.1016/j.isci.2022.105619

Figure Lengend Snippet:

Article Snippet: PC9 cells , RIKEN BioResource Research Center , RCB4455.

Techniques: Recombinant, Transfection, Library Quantification, Microarray, Control, Amplification, Sequencing, Cloning, Software, FACS, Digital PCR