Journal: Acta Biochimica et Biophysica Sinica

Article Title: LINC00891 regulated by miR-128-3p/GATA2 axis impedes lung cancer cell proliferation, invasion and EMT by inhibiting RhoA pathway

doi: 10.3724/abbs.2022005

Figure Lengend Snippet: LINC00891 regulates A549 and H460 cell functions via the RhoA pathway (A) A549 and H460 cells were transfected with LINC00891 overexpression vector, LINC00891 siRNA or respective negative controls, and the activated RhoA was determined using RhoA Activation Assay Kit. (B) Cells were treated with 15 μM CCG-1423 for 24 h, and the activated RhoA was assessed using RhoA Activation Assay Kit. Cells were treated with LINC00891 siRNA alone or together with 15 μM CCG-1423, and cell proliferation (C), invasion (D), and migration (E) were analyzed. (F) Western blot analysis was used to measure the protein levels of E-cadherin, Vimentin, Snail and Slug. n=5 in each group, *P<0.05, **P<0.01.

Article Snippet: RhoA activity was assessed using RhoA Activation Assay Kit (Merck Millipore, Billerica, USA) according to the manufacturer’s instructions.

Techniques: Transfection, Over Expression, Plasmid Preparation, Activation Assay, Migration, Western Blot

Journal: Neural Regeneration Research

Article Title: Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells

doi: 10.4103/1673-5374.239438

Figure Lengend Snippet: Effects of exogenous MAG treatment on RhoA activity in neuro-2a cells (ELISA assay). Data are expressed as the mean ± SE ( n = 6 per group; one-way analysis of variance followed by t -test). ** P < 0.01, *** P < 0.001. ELISA: Enzyme-linked immunosorbent assay; MAG: myelin-associated glycoprotein; ns: not significant; min: minutes.

Article Snippet: The activity of RhoA was measured using a commercial RhoA activity assay ELISA kit (Jingma Bioscientifics, Inc., Shanghai, China).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: EGFR inhibition leads to enhanced desmosome assembly and cardiomyocyte cohesion via ROCK activation

doi: 10.1172/jci.insight.163763

Figure Lengend Snippet: ( A ) RhoA G-LISA showing enhanced RhoA activity upon EGFR inhibition by erlotinib. * P ≤ 0.05, unpaired Student’s t test, n = 3. ( B ) Representative Western blot showing phosphorylation of the ROCK target MLC2 upon erlotinib treatment. * P ≤ 0.05, 2-way ANOVA with Holm Sidak’s multiple comparison test, n = 6 independent experiments. ( C ) Dispase-based dissociation assay in HL-1 cardiomyocytes, after treatment with erlotinib with and without Y27632. Y27632 was added 30 minutes prior to erlotinib incubation for 60 minutes, with representative pictures of the wells. * P ≤ 0.05, 2-way ANOVA with Holm Sidak’s multiple comparison test, n = 8 independent experiments. ( D ) Immunostaining of DP and DSG2 in HL-1 cardiomyocytes with WGA as membrane marker after erlotinib treatment with and without Y26732, as in C . White arrows indicate areas of increased DP or DSG2 recruitment to the cell membrane. Scale bar: 10 μm. ( E ) Dispase-based dissociation assay in HL-1 cardiomyocytes after 90 minutes of Ca 2+ depletion and treatment with erlotinib with and without Y26732 with representative pictures of the wells. * P ≤ 0.05, 2-way ANOVA with Holm-Sidak’s multiple comparison test, n = 7 independent experiments.

Article Snippet: RhoA G-LISA was performed using the RhoA G-LISA Activation Assay kit (Biozol, BK124), which measures cellular levels of GTP-loaded RhoA, according to the manufacturer’s instructions.

Techniques: Activity Assay, Inhibition, Western Blot, Phospho-proteomics, Comparison, Incubation, Immunostaining, Membrane, Marker

Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a KRAS Mut cell lines (H358, A427, NCIH727, NCIH23 and SKLU-1), EGFR Mut cell lines (HCC827, HCC2279, H1650 and H1975), KRAS WT and EGFR WT cell lines (H322M, H522, Calu-3 and HCC1666) and nontumorigenic cells (HEK-293T and BEAS-2B) were collected with lysis buffer, and SIRT1 activity was measured with cell lysates. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. b Normal human bronchial epithelial cell (BEAS-2B) and KRAS Mut cell lines used in a were collected with lysis buffer, and immunoblotted with anti-pMKK4, MKK, pMKK7, MKK, pJNK1, JNK1 and β-actin antibodies. c KRAS Mut cell lines (H358, A427 and NCIH727) were treated with anisomycin 38 μM (10 μg/ml) (JNK1 activator) and SP600125 20 μM (JNK1 inhibitor) for 2 h. The protein levels of pJNK1, JNK, pSIRT1 S27 , pSIRT1 S47 , pSIRT1 T530 , SIRT1 and β-actin were measured by western blot analysis. d H358 cells were treated with anisomycin and SP600125 under the same condition as in b and then whole-cell lysates were subjected to immunoprecipitation with an anti-JNK1 antibody. Immunoblot analysis was performed using antibodies against SIRT1, pSIRT1 S27 , pSIRT1 S47 , pSIRT1 T530 , pJNK1, JNK and β-actin antibodies. e The recombinant proteins, SIRT1 and JNK1 were incubated in the reaction mixture for phosphorylation at 32 °C for 4 h, and then Ser- and Thr-phosphorylated peptides were identified using anti-pSIRT1 S27 , pSIRT1 S47 , pSIRT1 T530 antibody. f H358 cells were transfected with SIRT1 WT , SIRT1 T530A , SIRT1 S27A&S47A , and then H358 cell extracts were immunoprecipitated with anti-KRAS antibody and RAF-1 agarose beads and immunoblotted with anti-acetylation, anti-SIRT1, anti-KRAS, anti-KRAS–GTP-bound and β-actin antibodies. g KRAS Mut cell lines (H358, A427 and H727) were transfected with SIRT1 WT , SIRT1 S27A , SIRT1 S47A , SIRT1 T530A and SIRT1 S27A,S47A (2 μg), then collected with lysis buffer, and SIRT1 activity was assessed with cell lysates. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05.

Article Snippet: The active GTP-bound KRAS was quantified using a KRAS activation assay kit (cat. no. STA-400-K, Cell Bio Labs) according to the manufacturer’s instructions.

Techniques: Lysis, Activity Assay, Western Blot, Immunoprecipitation, Recombinant, Incubation, Phospho-proteomics, Transfection

Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a Normal human bronchial epithelial cell (BEAS-2B) and KRAS Mut cell lines (H358, A427 and H727) were collected with lysis buffer and subjected to western blotting with anti-pERK, ERK and β-actin antibodies. b A luciferase assay was performed to assess the AP-1-mediated transcriptional regulatory activity with cell lysates in Fig. 4a. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. c TGFB1 mRNA expression was measured by RT–qPCR with same cell lines as in a . RPL32 was used as internal control and for normalization. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. d The medium of four cell lines were changed by FBS-free medium before cell collection at 24 h. Conditioned medium was collected and concentrated using an Amicon Ultra-15 tube, and total TGF-β1 levels were measured by ELISA. e KRAS Mut cell lines (H358, A427 and H727) were transfected with pcDNA , KRAS G12C , G12D and G12V plasmids (2 μg). TGF-β1 levels were measured under the same method as in d . f H358, A427 and H727 cells were transplanted with pcDNA , KRAS G12C , G12D and G12V plasmids, siCon and siSmad2/3 (80 nM) for 48 h, and then the activity of Smad2/3, JNK1 and KRAS was measured. g The cell lysates of each different KRAS Mut cell lines (H358, A427 and H727) under KWN-C with indicated dosage for 24 h were transferred by immunoblotting assay with anti-pSmad2/3, anti-Smad2/3, pJNK1, JNK1, anti-pSIRT1 Ser27 , pSIRT1 Ser47 , SIRT1, KRAS–GTP-bound and β-actin antibodies. h , H358, A427 and H460 cells were treated with DMSO or KWN-C (10 μM), and cell extracts were then immunoprecipitated using immunoglobulin G, anti-KRAS, and RAF-1 agarose bead antibodies. Immunoblotting was performed using anti-acetyl, anti-KRAS–GTP-bound, anti-SIRT1, anti-KRAS and β-actin antibodies.

Article Snippet: The active GTP-bound KRAS was quantified using a KRAS activation assay kit (cat. no. STA-400-K, Cell Bio Labs) according to the manufacturer’s instructions.

Techniques: Lysis, Western Blot, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Transfection, Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a H358, H460, NCIH23, SKLU-1 and SW900 cells were treated with CP (H358 1 μM, H460, NCIH23 and SKLU-1 5 μM), MTA (H358 10 μM, H460, NCIH23 and SKLU-1 5 μM) and/or KWN-C (10 μM), and cell proliferation was measured by the MTS assay 3 days after drug treatment. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. b Top: KRAS Mut cells were seeded with 0.5% top agar and cultured in a mixture of fresh medium with drugs as described in Supplementary Fig. . Cell colonies were stained with crystal violet and counted per 3.8 cm 2 . Bottom: representative colony images are shown. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. c H358 and H460 cells were treated with CP (H358 1 μM and H460 5 μM), MTA (H358 10 μM and H460 5 μM) and/or KWN-C (10 μM). Cell lysates from drug-treated cells were incubated with Raf-1–RBD to pull down KRAS–GTP (the active form of KRAS), followed by western blotting with an anti-KRAS antibody. Expression levels of KRAS, pERK, ERK, pAkt, Akt, PARP, cleaved PARP, pro-caspase-3, cleaved-caspase-3 and β-actin in total lysates were analyzed by western blotting. d H358 and H460 cells treated as in a were assessed for apoptosis by TUNEL assay (middle row), and their nuclei were stained with DAPI (top row; scale bar, 25 μm). All figures are representative of at least three separate experiments. e H358 and H460 cells treated as in a were stained with Annexin V/PI staining for apoptosis using flow cytometric analysis. Representative flow cytometry plots. All figures are representative of at least three separate experiments.

Article Snippet: The active GTP-bound KRAS was quantified using a KRAS activation assay kit (cat. no. STA-400-K, Cell Bio Labs) according to the manufacturer’s instructions.

Techniques: MTS Assay, Cell Culture, Staining, Incubation, Western Blot, Expressing, TUNEL Assay, Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer

doi: 10.1038/s12276-025-01536-8

Figure Lengend Snippet: a H358 cells harboring stably expressed luciferase plasmid were intratracheally injected into nude mice (1 × 10 6 cells per mouse). Top: representative bioluminescence images 2 months after the injection. The mice were euthanized 2 months after the injection, and lungs were excised and stained with Bouin’s fixative. Bottom: the lung tumor images. Therapeutic candidates were treated with CP (5 mg/kg per day, i.p.), MTA (150 mg/kg twice a week, i.p.) and/or KWN-C (30 mg/kg per day, i.p.). b The photon emission values represent the mean ± s.e.m. of the indicated number of mice. Student’s t -test, mean ± s.e.m.; n = 5, * P < 0.05. c The lung tumor weight from combination CP, MTA and/or KWN-C-treated mice was measured and compared with nontreatment, each single treatment and combined treatment. Student’s t -test, mean ± s.e.m.; n = 5, * P < 0.05. d The number of colonies formed in the lungs were measured under microscopy under the same conditions as in c . Student’s t -test, mean ± s.e.m.; n = 5, * P < 0.05. e KRAS–GTP (active form of KRAS) was pulled down by Raf-1–RBD from tumor tissue lysates, followed by western blot using KRAS antibody. Expression levels of anti-pSIRT1 S27 , pSIRT1 S47 , SIRT1, KRAS–GTP-bound, KRAS, pERK, ERK pAkt, Akt, PARP, cleaved PARP, pro-caspase-3, cleaved-caspase-3 and β-actin were analyzed by western blot in tumor tissues.

Article Snippet: The active GTP-bound KRAS was quantified using a KRAS activation assay kit (cat. no. STA-400-K, Cell Bio Labs) according to the manufacturer’s instructions.

Techniques: Stable Transfection, Luciferase, Plasmid Preparation, Injection, Staining, Microscopy, Western Blot, Expressing