Journal: Nature Communications

Article Title: Linker-free PROTACs efficiently induce the degradation of oncoproteins

doi: 10.1038/s41467-025-60107-7

Figure Lengend Snippet: A H3122 cells were incubated with Pro-BA, Pro-PEG1-BA and Pro-PEG3-BA separately at 100 nM for 48 h. The expression levels of EML4-ALK and GAPDH were assessed by immunoblotting. B H3122 cell viability was measured via CCK-8 assays following a 48-hour exposure to Pro-BA, Pro-PEG1-BA, and Pro-PEG3-BA at various concentrations. Data is represented as mean ± SD of three independent experiments. C Summary of DC 50 , D max , IC 50 values, and molecular weight (MW) for the indicated compounds. D H3122 cells were exposed to Pro-BA, Pro-PEG1-BA, or Pro-PEG3-BA at 500 nM for 24 h, and cell cycle distribution was assessed using flow cytometry. E The bar graph illustrates the percentage of H3122 cells in the G1, S, and G2 phases as shown in ( D ). F H3122 cells were treated with Pro-BA, Pro-PEG1-BA or Pro-PEG3-BA at 500 nM for 24 hours, and apoptosis was assessed using flow cytometry. G The bar graph shows the percentages of early (left) and late (right) apoptotic cells, as indicated by the Q3 and Q2 quadrants in ( F ), respectively. H H3122 cells were treated with 5 μM Pro-BA or Pro-PEG3-BA for 5 h. UHPLC-MS/MS analysis of intracellular amounts of Pro-BA and Pro-PEG3-BA per 5 × 10 6 cells. Data are presented as mean ± SD from three independent experiments, two-tailed Student’s t -test. For ( E ), ( G ), data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA followed by Fisher’s LSD test (two-tailed). The gating strategies for flow cytometry analysis are shown in Supplementary Fig. . Source data are provided as a file.

Article Snippet: The sections were blocked with goat serum for 30 min, followed by an overnight incubation at 4 °C with anti-ALK antibody (CST, 3633, 1:100 dilution).

Techniques: Incubation, Expressing, Western Blot, CCK-8 Assay, Molecular Weight, Flow Cytometry, Tandem Mass Spectroscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Linker-free PROTACs efficiently induce the degradation of oncoproteins

doi: 10.1038/s41467-025-60107-7

Figure Lengend Snippet: A H3122 cells were treated with Pro-BA alone or in combination with MG132 (10 µM) or CQ (25 µM), for 6 h. The protein levels of EML4-ALK and GAPDH were subsequently assessed by immunoblotting (upper panel). The bar graph presents a quantitative analysis of EML4-ALK protein levels derived from three independent replicates shown in Fig. 4A and Supplementary Fig. (lower panel). B H3122 cells were treated with cycloheximide (CHX) alone, or in combination with Pro-BA, or with both Pro-BA (100 nM) and MG132 (10 µM) for different time intervals, followed by immunoblotting to measure EML4-ALK and GAPDH expression (upper panel). Quantitative analysis of the Western blot results for EML4-ALK from three separate repeats is presented in Fig. 4B and Supplementary Fig. (lower panel). C Immunoblot analysis was conducted on H3122 cells pre-treated with BA (10 μM) for 2 h and then incubated with Pro-BA (100 nM) for 12 h to assess the expression of specific proteins (upper panel). The bar graph presents a quantitative analysis of EML4-ALK protein levels derived from three independent experiments shown in Fig. 4C and Supplementary Fig. (lower panel). D H3122 cells with stable expression of the indicated sgRNA were exposed to either DMSO or Pro-BA for 24 h, and the levels of the indicated protein were assessed by immunoblotting. E HEK293T cells co-expressing HA-GID4 and Flag-EML4-ALK were treated with DMSO, Pro-BA (500 nM), or Brigatinib (500 nM) for 24 h. The cells were then subjected to immunoprecipitation using anti-FLAG® M2 Magnetic Beads, followed by immunoblotting with the specified antibodies. F HEK293T cells coexpressing HA-GID4, Flag-EML4-ALK, and myc-Ub were incubated with DMSO, Pro-BA (500 nM), or Brigatinib (500 nM) for 24 h, then treated with MG132 (10 μM) for 4 h. Ubiquitylation of Flag-EML4-ALK was analyzed by denaturing immunoprecipitation (IP) with an anti-myc-tag antibody. G ITC measurement of the affinity of Pro-BA (left) and Pro-PEG3-BA (right) with ALK (1094-1400 aa). H HEK293T cells co-transfected with pHTN-GID4 and pNLF1-N-ALK were exposed to Brigatinib, Pro-BA, or Pro-PEG3-BA at the indicated concentration for 6 h. Data represented as normalized NanoBRET ratio. Data are presented as the mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA followed by Fisher’s LSD test (two-tailed) for pairwise comparisons. I HEK293T cells coexpressing HA-GID4 and Flag-EML4-ALK were incubated with DMSO, Pro-BA (500 nM), Pro-PEG3-BA (500 nM), or Brigatinib (500 nM) for 24 h, respectively. After immunoprecipitation with anti-FLAG® M2 Magnetic Beads, followed by immunoblotting with various antibodies indicated. J HEK293T cells coexpressing HA-GID4, Flag-EML4-ALK, and myc-Ub were treated with DMSO, Pro-BA (500 nM), Pro-PEG-BA (500 nM), or Brigatinib (500 nM) for 24 h, followed by the addition of MG132 (10 μM) for 4 h. Ubiquitination of Flag-EML4-ALK was examined by denaturing immunoprecipitation (IP) with anti-myc-tag antibody. For ( A – C ), data are shown as the mean ± SD ( n = 3 independent experiments), two-tailed Student’s t -test. Source data are provided as a file.

Article Snippet: The sections were blocked with goat serum for 30 min, followed by an overnight incubation at 4 °C with anti-ALK antibody (CST, 3633, 1:100 dilution).

Techniques: Western Blot, Derivative Assay, Expressing, Incubation, Immunoprecipitation, Magnetic Beads, Transfection, Concentration Assay, Two Tailed Test, Ubiquitin Proteomics

Journal: Signal Transduction and Targeted Therapy

Article Title: RNase1-driven ALK-activation is an oncogenic driver and therapeutic target in non-small cell lung cancer

doi: 10.1038/s41392-025-02206-x

Figure Lengend Snippet: RNase1 binds to and activates ALK as its ligand in lung cancer cells. a Each of the 13 recombinantly purified 6´ N-terminal His-tagged RNases (10 µg) was incubated with H1299 lysate followed by Ni-His beads pull down and Western blot analysis with the indicated antibodies. b Each of the 13 RNases was added into the H1299 culture medium at a final concentration of 1 mg/ml. After 30 min incubation, cells were lysed and subjected to Western blotting with the indicated antibodies. A phospho-ALK antibody was used to detect ALK Y1604 phosphorylation. c Western blot analysis of ALK phosphorylation sites in HEK293 cells. Flag-tagged wild type or Tyr mutant ALK plasmids were transfected into HEK293 cells and then IP with Flag-beads. A pan-phosphorylated Tyr antibody (4G10) was used to detect ALK phosphorylation. d Plasmids expressing Myc-tagged ALK and C-terminal Flag-tagged RNase1 (R1) were transfected into HeLa cells. Cell lysates were harvested and subjected to co-immunoprecipitation (co-IP) assay. RNase1 was detected using the Flag antibody. RNase5 (R5) was used as a negative control. e In vitro binding affinity assay of ALK and RNase1. Kd, dissociation constant. BSA was used as a negative control. f Immunofluorescence microscopy of H1299 cells with or without RNase1 expression. Yellow dots and white arrows both indicate co-localization of ALK and RNase1. Scale bar, 20 mm. g H1299 cells with or without RNase1 expression were subjected to Duo Link assay. Red dots indicate binding between ALK and RNase1. Scale bar, 20 mm. h Time course analysis of ALK activation in H1299 cells by RNase1 (1 mg/ml). ALK phos-Y1604 was used as an indicator of ALK activation. i Co-IP of ALK and RNAse1 from HEK293T cells transfected with wild-type (WT) or catalytic-deficient (CD) RNase1-expressing plasmid followed by Western blotting with the indicated antibodies

Article Snippet: Antibodies against RNase1 (Sigma HPA001140), ALK (Cell Signaling 3633), phosphorylated ALK (Tyr1604; Cell Signaling 3341S), phosphorylated ERK1/2 (Thr202/Tyr204; Cell Signaling 9101), ERK1/2 (Cell Signaling 9102), STAT3 (Cell Signaling 9132), phosphorylated STAT 3 (Cell Signaling 9145S), PD-L1 (Cell Signaling 13684 s), and tubulin (Sigma B-5-1-2) were used for immunoblotting, immunohistochemistry, immunofluorescence, and Duolink assays.

Techniques: Purification, Incubation, Western Blot, Concentration Assay, Phospho-proteomics, Mutagenesis, Transfection, Expressing, Co-Immunoprecipitation Assay, Negative Control, In Vitro, Binding Assay, Immunofluorescence, Microscopy, Activation Assay, Plasmid Preparation

Journal: Signal Transduction and Targeted Therapy

Article Title: RNase1-driven ALK-activation is an oncogenic driver and therapeutic target in non-small cell lung cancer

doi: 10.1038/s41392-025-02206-x

Figure Lengend Snippet: Identification of patients with RDAA NSCLC. a Quantification of plasma RNase1 in NSCLC patients (N = 48) and normal individuals (N = 15). RNase1 concentration was measured by ELISA as described in Methods. p < 0.01, Student’s t -test. b IHC staining of RNase1 in human lung tumors (N = 48) and normal lung (N = 10) tissues. RNase1 expression level was calculated based on the intensity and percentage of stained cells as described in Methods. Representative images shown. c Correlation analysis between plasma RNase1 concentration and RNase1 expression level in paired tumor tissues (N = 47). R = 0.84, Pearson’s Chi-Square test. d IHC staining of RNase1 expression and ALK phosphorylation levels in human NSCLC tissues. ALK p-Y1604, ALK p-Y1282/1283 and RNase1 specific antibodies were used for IHC staining. Representative images shown. e Diagnosis of RDAA NSCLC patients in 1173 NSCLC tissues. ALK p-Y1604, ALK p-Y1282/1283 and RNase1 were used as biomarkers to identify RDAA positive samples. f Correlation analysis between RNase1 expression and ALK phosphorylation in NSCLC tissues which used from ( e ). ALK p-Y1604 and 1282/1283 double positive means ALK phosphorylation positive (pALK + ), otherwise means ALK phosphorylation negative (pALK-)

Article Snippet: Antibodies against RNase1 (Sigma HPA001140), ALK (Cell Signaling 3633), phosphorylated ALK (Tyr1604; Cell Signaling 3341S), phosphorylated ERK1/2 (Thr202/Tyr204; Cell Signaling 9101), ERK1/2 (Cell Signaling 9102), STAT3 (Cell Signaling 9132), phosphorylated STAT 3 (Cell Signaling 9145S), PD-L1 (Cell Signaling 13684 s), and tubulin (Sigma B-5-1-2) were used for immunoblotting, immunohistochemistry, immunofluorescence, and Duolink assays.

Techniques: Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Expressing, Staining, Phospho-proteomics, Biomarker Discovery

Journal: Signal Transduction and Targeted Therapy

Article Title: RNase1-driven ALK-activation is an oncogenic driver and therapeutic target in non-small cell lung cancer

doi: 10.1038/s41392-025-02206-x

Figure Lengend Snippet: RDAA is an oncogenic driver in vitro and in vivo. a Western blot analysis of H1299 stable cells expressing wild-type RNase1 and/or short hairpin RNA (shRNA) to knockdown ALK with the indicated antibodies. b The indicated H1299 cells were subjected to MTT assay and cell viability was quantified. ** p < 0.01, Student’s t -test. c Cell counting of the indicated cells was performed in triplicate and normalized to control. Error bars, mean ± SD. ** p < 0.01. d Colony formation assay of the indicated cells. The relative number of colonies formed was measured in triplicate. ** p < 0.01. e Wound healing assay of the indicated cells. Representative images shown. Scale bar, 500 mm. Quantification shown in Supplementary Fig. . f Nude mice were injected with NIH3T3 cells or those expressing ALK and/or RNase 1. Tumor size was measured every 3 days. Red arrows pointing to tumors developed. g Quantification of tumors in ( f ). N = 8, control, R1, ALK and R1-ALK co-expression group. N = 5, ALK mutant group. ** p < 0.01, Student’s t -test. NS, not significant. h Western blo t analysis of ALK-RNase1 tumors (N = 7). Actin was used as control. i Correlation between serum RNase1 concentration and tumor size. R = 0.89, Pearson’s Chi-Square test. j Western blot analysis of NIH3T3 cells with ALK and RNase1 high or RNase1 low expression. k Quantification of tumor size from mice injected with RNase1 high or RNase1 low NIH3T3 cells. N = 5. * p < 0.05, Student’s t test

Article Snippet: Antibodies against RNase1 (Sigma HPA001140), ALK (Cell Signaling 3633), phosphorylated ALK (Tyr1604; Cell Signaling 3341S), phosphorylated ERK1/2 (Thr202/Tyr204; Cell Signaling 9101), ERK1/2 (Cell Signaling 9102), STAT3 (Cell Signaling 9132), phosphorylated STAT 3 (Cell Signaling 9145S), PD-L1 (Cell Signaling 13684 s), and tubulin (Sigma B-5-1-2) were used for immunoblotting, immunohistochemistry, immunofluorescence, and Duolink assays.

Techniques: In Vitro, In Vivo, Western Blot, Expressing, shRNA, Knockdown, MTT Assay, Cell Counting, Control, Colony Assay, Wound Healing Assay, Injection, Mutagenesis, Concentration Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: RNase1-driven ALK-activation is an oncogenic driver and therapeutic target in non-small cell lung cancer

doi: 10.1038/s41392-025-02206-x

Figure Lengend Snippet: RDAA tumors are sensitive to ALK inhibitor in both subcutaneous and orthotopic mouse models. a Western blot analysis of ALK downstream signaling of RNase1-driven ALK-positive (RDAA) H1299 cells with the indicated antibody. Phospho-ERK1/2 T202/Y204 and phospho-STAT3 (Y705) specific antibodies were used to detect ERK1/2 and STAT3 phosphorylation, respectively. b RNA-seq analysis of control, RDAA and ALK -rearranged (EML4-ALK) H2228 lung cancer cells. c Cell counting of the indicated cells treated with or without (DMSO; control) the indicated ALK inhibitors. Experiments were performed in triplicate. d Mice received the indicate H1299 cells by subcutaneous injection, and when tumors reached 500 mm 3 , ALK inhibitor Ceritinib (25 mg/kg/d) was administered beginning day 14 for 2 weeks. Tumor size was measured every 3 days. N = 10. e Overall survival curve of mice in ( d ) starting the day of cell injection. f Western blot analysis of ALK phosphorylation with or without Ceritinib treatment. The tumor samples were collected from the mice in ( e ). g Representative Micro-CT scan images of mice who received an orthotopic injection of the indicated cells treated with or without Ceritinib (25 mg/kg/d). N = 7. Red arrows pointing to tumors. h Overall survival curve of mice in ( e ) starting the day of tumor transplantation. ** p < 0.01, Log-rank (Mantel-Cox) test. i Representative 3D images of mice with orthotopic RDAA lung tumor before or after Ceritinib treatment. Area in pink indicate tumor size and location

Article Snippet: Antibodies against RNase1 (Sigma HPA001140), ALK (Cell Signaling 3633), phosphorylated ALK (Tyr1604; Cell Signaling 3341S), phosphorylated ERK1/2 (Thr202/Tyr204; Cell Signaling 9101), ERK1/2 (Cell Signaling 9102), STAT3 (Cell Signaling 9132), phosphorylated STAT 3 (Cell Signaling 9145S), PD-L1 (Cell Signaling 13684 s), and tubulin (Sigma B-5-1-2) were used for immunoblotting, immunohistochemistry, immunofluorescence, and Duolink assays.

Techniques: Western Blot, Phospho-proteomics, RNA Sequencing, Control, Cell Counting, Injection, Micro-CT, Transplantation Assay