Journal: Oncology Reports

Article Title: Inhibition of primary ciliogenesis enhances efficacy of EGFR-TKIs against non-small cell lung cancer cells

doi: 10.3892/or.2025.9035

Figure Lengend Snippet: Epidermal growth factor receptor-tyrosine kinase inhibitor-insensitive A549 cells harbor high cilia incidence. (A and B) A549 cells were treated with increasing concentrations of (A) Gef or (B) Dac, and the cell viability related to untreated control (Ctrl) was measured at 48 h post-treatment. (C and D) HCC827 cells were treated with increasing concentrations of (C) Gef or (D) Dac, and the cell viability related to untreated control was measured at 48 h post-treatment. (E and F) A549 and HCC827 cells were exposed to increasing concentrations of Gef for 48 h, and the immunoblotting analysis was performed to measure the protein expression levels of ERK and p-ERK at Thr202/Tyr204. α-tubulin (α-tub) was used as a loading control. (G) Representative images of primary cilia labeled with ARL13B (red) and basal bodies labeled with γ-tub (green) in A549 and HCC827 cells, and the cilia incidence was calculated. The nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. All quantitative data were obtained from three replicates and shown as the mean ± SD. Error bars, ± SD. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 compared with 0 µM (0.1% DMSO). Gef, gefitinib; Dac, dacomitinib; p-, phosphorylated; SD, standard deviation.

Article Snippet: The first and second generation of EGFR-TKIs gefitinib (Gef; cat. no. HY-50895) and dacomitinib (Dac; cat. no. HY-13272) were purchased from MedChemExpress.

Techniques: Control, Western Blot, Expressing, Labeling, Standard Deviation

Journal: Oncology Reports

Article Title: Inhibition of primary ciliogenesis enhances efficacy of EGFR-TKIs against non-small cell lung cancer cells

doi: 10.3892/or.2025.9035

Figure Lengend Snippet: Epidermal growth factor receptor-tyrosine kinase inhibitors promote ciliogenesis in dose-dependent manner in A549 but not HCC827 cells. (A) Representative images of primary cilia labeled with ARL13B (red) and basal bodies labeled with γ-tub (green) in A549 cells treated with increasing doses of Gef or Dac for 48 h. (B and C) Quantification of (B) ciliated cells and the (C) cilium length of A549 cells treated with 0 (n=117), 1 (n=176), 5 (n=133), 10 (n=216), 25 (n=210), 50 (n=95) µM Gef for 48 h. **P<0.01 and ****P<0.0001 compared with 0 µM; ns, not significant. (D and E) Quantification of (D) ciliated cells and the (E) cilium length of A549 cells treated with 0 (n=133), 0.5 (n=153), 1 (n=138), 5 (n=163), 7.5 (n=130), 10 (n=80) µM Dac for 48 h. *P<0.05 and ****P<0.0001 compared with 0 µM; ns, not significant. (F) Representative images of primary cilia labeled with ARL13B (red) and basal bodies labeled with γ-tub (green) in HCC827 cells treated with 0.01 µM Gef or 0.001 µM Dac for 48 h. (G) Quantification of ciliated cells in HCC827 cells in (F). ns, not significant compared with DMSO. The nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. All quantitative data were obtained from three replicates and shown as the mean ± SD. Error bars, ± SD. Gef, gefitinib; Dac, dacomitinib; p-, phosphorylated; SD, standard deviation; ns, not significant.

Article Snippet: The first and second generation of EGFR-TKIs gefitinib (Gef; cat. no. HY-50895) and dacomitinib (Dac; cat. no. HY-13272) were purchased from MedChemExpress.

Techniques: Labeling, Standard Deviation

Journal: Oncology Reports

Article Title: Inhibition of primary ciliogenesis enhances efficacy of EGFR-TKIs against non-small cell lung cancer cells

doi: 10.3892/or.2025.9035

Figure Lengend Snippet: Gef treatment leads to G1 phase arrest and senescence. (A and B) Cell cycle distribution assay of A549 cells treated with DMSO (0.1%) or 25 µM Gef for 1, 2, and 3 days (d) by flow cytometry with (A) PI staining; (B) quantification data. (C and D) Cell cycle distribution assay of HCC827 cells treated with DMSO (0.1%) or 0.05 µM Gef for 1, 2, and 3 days by flow cytometry with (C) PI staining; (D) quantification data. (E) Immunoblotting analysis of the expression levels of CCNA, CCNB, CCND, CCNE, CDK1, CDK2, p16 and p21 in A549 cells treated with DMSO, Gef (25 µM), or Dac (5 µM) for 1 and 3 days. α-tubulin (α-tub) and GAPDH were used as loading controls. (F) Representative PI staining images of A549 cells exposed to DMSO (0.1%) or Gef (25 µM) for 1, 2, 3 days, the dead cells (red) were stained with PI and indicated with red arrows. Scale bar, 100 µm. (G) Immunoblotting analysis of the expression levels of IFT88, p-ERK (Thr202/Tyr204) and BCL2 in A549 cells treated with DMSO (0.1%) or Gef (25 µM) for 1, 2, 3 days. α-tubulin (α-tub) was used as a loading control. The values under the immunoblot bands indicate the quantitative densitometry value measured using ImageJ software. (H and I) The (H) cellular senescence assay of A549 cells treated with DMSO (0.1%) or Gef (25 µM) for 1–3 d labeling by senescence-tracker (Sen-Tra) fluorescence probe, and the (I) quantification data. Scale bar, 100 µm. Data are expressed as the mean ± SD. Error bars, ± SD. *P<0.05, **P<0.01 and ***P<0.001 compared with DMSO. Gef, gefitinib; PI, propidium iodide; CCNA, Cyclin A2; CCNB, Cyclin B1; CCND, Cyclin D1; CCNE, Cyclin E1; Dac, dacomitinib; BF, bright field; p-, phosphorylated; SD, standard deviation.

Article Snippet: The first and second generation of EGFR-TKIs gefitinib (Gef; cat. no. HY-50895) and dacomitinib (Dac; cat. no. HY-13272) were purchased from MedChemExpress.

Techniques: Flow Cytometry, Staining, Western Blot, Expressing, Control, Software, Labeling, Fluorescence, Standard Deviation

Journal: Oncology Reports

Article Title: Inhibition of primary ciliogenesis enhances efficacy of EGFR-TKIs against non-small cell lung cancer cells

doi: 10.3892/or.2025.9035

Figure Lengend Snippet: Inhibition of ciliogenesis sensitizes A549 cells to epidermal growth factor receptor-tyrosine kinase inhibitors. (A) Immunoblotting analysis of the protein expression levels of IFT88 and ARL13B in A549 cells transfected with siRNAs against IFT88 (siIFT88-1/2), ARL13B (siARL13B-1/2), or NC. GAPDH was employed as a loading control. The values under the immunoblot bands indicate the quantitative densitometry value measured using ImageJ software. (B-D) Immunofluorescence labeling of primary cilia (ARL13B, red) and basal bodies (γ-tub, green) in A549 control cells (Ctrl) and cells transfected with siIFT88-1/2, siARL13B-1/2, or NC following treated with 10 µM Gef for 48 h, (C) the proportion of ciliated cells and (D) cilium length in each group. Ctrl, n=52; Gef-NC, n=59; Gef-siIFT88-1, n=57; Gef-siIFT88-2, n=64; Gef-siARL13B-1, n=56; Gef-siARL13B-2, n=69. The nuclei were stained with DAPI (blue). (E and F) A549 cells transfected with NC, siIFT88-1/2, or ARL13B-1/2 siRNAs were exposed to (E) Gef (0, 5, 10 µM) or (F) Dac (0, 1, 5 µM) for 48 h, the cell viability related to untreated control was measured. (G and H) A549 cells transfected with indicated siRNAs were exposed 10 µM Gef for 48 h and stained with PI to detect the dead cells and quantification data for each group. Scale bar, 100 µm. Data are expressed as the mean ± SD. Error bars, ± SD. *P<0.05, **P<0.01 and ***P<0.001 compared with NC. siRNA, small interfering RNA; NC, negative control; Gef, gefitinib; Dac, dacomitinib; BF, bright field; SD, standard deviation.

Article Snippet: The first and second generation of EGFR-TKIs gefitinib (Gef; cat. no. HY-50895) and dacomitinib (Dac; cat. no. HY-13272) were purchased from MedChemExpress.

Techniques: Inhibition, Western Blot, Expressing, Transfection, Control, Software, Immunofluorescence, Labeling, Staining, Small Interfering RNA, Negative Control, Standard Deviation

Journal: Oncology Reports

Article Title: Inhibition of primary ciliogenesis enhances efficacy of EGFR-TKIs against non-small cell lung cancer cells

doi: 10.3892/or.2025.9035

Figure Lengend Snippet: Epidermal growth factor receptor-tyrosine kinase inhibitors induce AC3 expression and ciliary localization. (A) Immunoblotting analysis of the expression levels of AC3 and ARL13B in A549 cells exposed to DMSO (0.1%), Gef (10 µM) and Dac (5 µM) for 1 and 3 days. (B and C) Immunofluorescence labeling of primary cilia (Arl13b, green) and AC3 (red, indicated by white arrows) in A549 cells administrated with DMSO (0.1%), Gef (10 µM), and Dac (5 µM) for 3 days, and quantification of AC3 positive (AC3 + ) cilia in each group. The nuclei were stained with DAPI (blue). Scale bar, 10 µm. Data are expressed as the mean ± SD. Error bars, ± SD. ***P<0.001 compared with DMSO. (D) Immunoblotting analysis of the expression levels of AC3 and ARL13B in HCC827 cells exposed to DMSO (0.1%), Gef (0.05 µM) and Dac (0.01 µM) for 3 days. (E) Immunoblotting analysis of the protein expression levels of AC3 and IFT88 in Gef-treated A549 cells transfected with siIFT88-1 (siIFT88) or NC siRNA for 3 days. (F) Immunofluorescence labeling of primary cilia (Arl13b, red) and AC3 (green) in cells in (E). Scale bar, 5 µm. GAPDH was used as a loading control; the values under the immunoblot bands indicate the quantitative densitometry value measured using ImageJ software. Gef, gefitinib; Dac, dacomitinib; SD, standard deviation; si-, small interfering; NC, negative control; AC3, adenylate cyclase 3.

Article Snippet: The first and second generation of EGFR-TKIs gefitinib (Gef; cat. no. HY-50895) and dacomitinib (Dac; cat. no. HY-13272) were purchased from MedChemExpress.

Techniques: Expressing, Western Blot, Immunofluorescence, Labeling, Staining, Transfection, Control, Software, Standard Deviation, Negative Control

Journal: Oncology Reports

Article Title: Inhibition of primary ciliogenesis enhances efficacy of EGFR-TKIs against non-small cell lung cancer cells

doi: 10.3892/or.2025.9035

Figure Lengend Snippet: Silencing AC3 expression enhances cellular sensitivity to Gef. (A) Immunoblotting analysis of the expression levels of AC3 and ARL13B in A549 cells transfected with siRNAs targeting AC3 (siAC3-1/2/3) or NC for 48 h. GAPDH was used as a loading control. The values under the immunoblot bands indicate the quantitative densitometry value measured using ImageJ software. (B) A549 cells transfected NC or siAC3-1/2/3 were exposed to Gef (0, 5, 10 µM) for 48 h, the cell viability related to untreated control was measured. (C and D) Immunofluorescence labeling of primary cilia (Arl13b, red, indicated by white arrows) in A549 cells transfected with NC and siAC3-1 following administrated with Gef (10 µM) for 3 days, and quantification of ciliated cells in each group. The nuclei were stained with DAPI (blue). Scale bar, 10 µm. Data are expressed as the mean ± SD. Error bars, ± SD. *P<0.05 and **P<0.01 compared with NC. AC3, adenylate cyclase 3; Gef, gefitinib; si-, small interfering; NC, negative control; SD, standard deviation; ns, not significant.

Article Snippet: The first and second generation of EGFR-TKIs gefitinib (Gef; cat. no. HY-50895) and dacomitinib (Dac; cat. no. HY-13272) were purchased from MedChemExpress.

Techniques: Expressing, Western Blot, Transfection, Control, Software, Immunofluorescence, Labeling, Staining, Negative Control, Standard Deviation

Journal: Cancer Discovery

Article Title: Sevabertinib, a Reversible HER2 Inhibitor with Activity in Lung Cancer

doi: 10.1158/2159-8290.CD-25-0605

Figure Lengend Snippet: Sevabertinib shows potent antiproliferative activity in isogenic Ba/F3 cells ectopically expressing HER2 exon 20 insertions or HER2 missense mutations. A, HER2 exon 20 insertions and single-nucleotide HER2 missense mutations in the extracellular domain, transmembrane domain (TM), and kinase domain of HER2 protein. B, Dose–response curves of sevabertinib in isogenic Ba/F3 cell lines ectopically expressing HER2 exon 20 insertions. C, IC 50 values of sevabertinib and the EGFR/HER2 TKIs zongertinib, tucatinib, lapatinib, and neratinib in isogenic Ba/F3 cell lines ectopically expressing HER2 missense mutations. D, Dose–response curves of sevabertinib in isogenic Ba/F3 cell lines ectopically expressing HER2 missense mutations. E, IC 50 values of sevabertinib and the EGFR/HER2 TKIs zongertinib, tucatinib, lapatinib, and neratinib in isogenic Ba/F3 cell lines ectopically expressing HER2 missense mutations. Dose–response curves of sevabertinib in isogenic Ba/F3 cell lines ectopically expressing HER2 exon 20 insertions (A775insYVMA, P780insGSP) or point mutation (S310F) coupled with ( F ) HER2 C805S mutation or ( G ) HER2 T798I or T798M mutations, predicted to cause TKI resistance. Ba/F3 cells transduced with an empty vector and maintained in the presence of IL3 were included as a control.

Article Snippet: EGFR/HER2 TKIs tucatinib, lapatinib, neratinib, and zongertinib were purchased from Selleck Chemicals, and STX-721 from MedChemExpress.

Techniques: Activity Assay, Expressing, Mutagenesis, Transduction, Plasmid Preparation, Control

Journal: Cancer Discovery

Article Title: Sevabertinib, a Reversible HER2 Inhibitor with Activity in Lung Cancer

doi: 10.1158/2159-8290.CD-25-0605

Figure Lengend Snippet: Sevabertinib shows potent antiproliferative activity in isogenic Ba/F3 cells ectopically expressing HER2 exon 20 insertions or HER2 missense mutations. A, HER2 exon 20 insertions and single-nucleotide HER2 missense mutations in the extracellular domain, transmembrane domain (TM), and kinase domain of HER2 protein. B, Dose–response curves of sevabertinib in isogenic Ba/F3 cell lines ectopically expressing HER2 exon 20 insertions. C, IC 50 values of sevabertinib and the EGFR/HER2 TKIs zongertinib, tucatinib, lapatinib, and neratinib in isogenic Ba/F3 cell lines ectopically expressing HER2 missense mutations. D, Dose–response curves of sevabertinib in isogenic Ba/F3 cell lines ectopically expressing HER2 missense mutations. E, IC 50 values of sevabertinib and the EGFR/HER2 TKIs zongertinib, tucatinib, lapatinib, and neratinib in isogenic Ba/F3 cell lines ectopically expressing HER2 missense mutations. Dose–response curves of sevabertinib in isogenic Ba/F3 cell lines ectopically expressing HER2 exon 20 insertions (A775insYVMA, P780insGSP) or point mutation (S310F) coupled with ( F ) HER2 C805S mutation or ( G ) HER2 T798I or T798M mutations, predicted to cause TKI resistance. Ba/F3 cells transduced with an empty vector and maintained in the presence of IL3 were included as a control.

Article Snippet: EGFR/HER2 TKIs tucatinib, lapatinib, neratinib, and zongertinib were purchased from Selleck Chemicals, and STX-721 from MedChemExpress.

Techniques: Activity Assay, Expressing, Mutagenesis, Transduction, Plasmid Preparation, Control

Journal: EMBO Molecular Medicine

Article Title: TKI-mediated inhibition of NLRP1 inflammasome restores erythropoiesis in DBA syndrome

doi: 10.1038/s44321-025-00368-3

Figure Lengend Snippet: ( A ) K562 cells were pretreated with 0.1 μM nilotinib, followed by differentiation with 50 μM hemin for 24 h before transcriptomic analysis. ( B ) Heatmap showing the top upregulated erythroid genes upon nilotinib treatment. ( C–E ) RT–qPCR analysis of select upregulated erythroid genes involved in structural components of erythrocytes ( C ), heme biosynthesis ( D ), and other erythropoietic functions ( E ) following nilotinib treatment. Data are shown as the mean ± SEM ( N = 3). P values were calculated using one-way ANOVA and Tukey’s multiple range test, ns, non-significant; ** p < 0.01, *** P < 0.001 and **** P < 0.0001. ( C ) ALAD: 0_DMSO wrt 0_NILO p = 0.0296*, 0_NILO wrt 24_NILO p = 0.0158*, FECH: 0_DMSO wrt 0_NILO p = 0.0451*, 0_NILO wrt 24_NILO p = 0.0405*, HMBS: 0_NILO wrt 24_NILO p = 0.0294*; ( D ) GYPC 0_NILO wrt 24_NILO p = 0.0096**, HBA1: 0_DMSO wrt 0_NILO p = 0.0193*, 0_NILO wrt 24_NILO p = 0.0418*, HBA2: 0_DMSO wrt 0_NILO p = 0.0107*, 0_NILO wrt 24_NILO p = 0.0287*; ( E ) SOD3: 0_DMSO wrt 0_NILO p = 0.0128*, 0_NILO wrt 24_NILO p = 0.0273*, NFE2: 0_DMSO wrt 24_DMSO p = 0.0465*, 0_NILO wrt 24_NILO p = 0.0497*. .

Article Snippet: One dpf larvae were manually dechorionated at 24 hpf and treated for 24 h by bath immersion with the TKIs nilotinib (#HY-10159, 1 μM), imatinib (#HY-15463, 1 μM and 10 μM), dasatinib (#HY-10181, 0.1 μM and 1 μM), bosutinib (#HY-10158, 0.1 μM and 1 μM) and ponatinib (#HY-12047, 0.1 μM and 1 μM), all from MedChemExpress, diluted in egg water supplemented with 0.1% DMSO.

Techniques: Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: TKI-mediated inhibition of NLRP1 inflammasome restores erythropoiesis in DBA syndrome

doi: 10.1038/s44321-025-00368-3

Figure Lengend Snippet: ( A , E ) Primary human CD34 + /CD133 + HSPCs from healthy donors were cultured with EPO, and 0.1 µM nilotinib was added from days 3 to 7 ( A ) or days 7 to 11 ( E ). ( B , F ) CASP1 activity was measured by flow cytometry with FAM FLICA and normalized to untreated control cells. ( D , G ) Erythroid differentiation was assessed by flow cytometry after staining with anti-CD235A-APC (Glycophorin A) and anti-CD71-FITC (Transferrin Receptor). Representative dot plots of differentiation stages are shown. The differentiation score was calculated as the ratio between CD235A + /CD71 + (intermediate erythroid progenitors) and CD235A - /CD71 + (early erythroid progenitors). MFI, mean fluorescence intensity. Data are shown as the mean ± SEM ( B , F : N = 3; D : N = 5, G : N = 2). P values were calculated using one-way ANOVA and Tukey’s multiple range test. ns, non-significant; ** p < 0.01, *** P < 0.001 and **** P < 0.0001. ( B ) DMSO_D7 wrt NILO_D7 p < 0.0001****, ( D ) DMSO_D5 wrt NILO_D5 p = 0.002***, DMSO_D7 wrt NILO_D7 p < 0.0001****, ( F ) DMSO_D10 wrt NILO_D10 p = 0.0005***. .

Article Snippet: One dpf larvae were manually dechorionated at 24 hpf and treated for 24 h by bath immersion with the TKIs nilotinib (#HY-10159, 1 μM), imatinib (#HY-15463, 1 μM and 10 μM), dasatinib (#HY-10181, 0.1 μM and 1 μM), bosutinib (#HY-10158, 0.1 μM and 1 μM) and ponatinib (#HY-12047, 0.1 μM and 1 μM), all from MedChemExpress, diluted in egg water supplemented with 0.1% DMSO.

Techniques: Cell Culture, Activity Assay, Flow Cytometry, Control, Staining, Fluorescence

Journal: EMBO Molecular Medicine

Article Title: TKI-mediated inhibition of NLRP1 inflammasome restores erythropoiesis in DBA syndrome

doi: 10.1038/s44321-025-00368-3

Figure Lengend Snippet: ( A ) Primary human CD34 + cells were purified from human cord blood and differentiated for 7 days with EPO in the presence of either DMSO, 100 nM nilotinib, 100 nM imatinib or 1 nM dasatinib. ( B , C ) Cells were stained with anti-GATA1-APC and GATA1 high cells analyzed by flow cytometry at 3 and 7 days post-differentation. Representative dot plots at different differentiation times are also shown. Data are shown as the mean ± SEM. P values were calculated using one-way ANOVA and Tukey’s multiple range test. All the significant comparisons have a p < 0.0001****. .

Article Snippet: One dpf larvae were manually dechorionated at 24 hpf and treated for 24 h by bath immersion with the TKIs nilotinib (#HY-10159, 1 μM), imatinib (#HY-15463, 1 μM and 10 μM), dasatinib (#HY-10181, 0.1 μM and 1 μM), bosutinib (#HY-10158, 0.1 μM and 1 μM) and ponatinib (#HY-12047, 0.1 μM and 1 μM), all from MedChemExpress, diluted in egg water supplemented with 0.1% DMSO.

Techniques: Purification, Staining, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: TKI-mediated inhibition of NLRP1 inflammasome restores erythropoiesis in DBA syndrome

doi: 10.1038/s44321-025-00368-3

Figure Lengend Snippet: ( A ) Schematic representation of experimental design. Zebrafish larvae were treated with the indicated concentrations of different TKIs from 1 to 7 days post-fertilization (dpf). ( B ) Survival rates of zebrafish larvae after TKI treatment at the specified doses, monitored from 1 to 7 dpf. ( C – E ) Effects of TKIs on hematopoiesis in zebrafish larvae. Caspase-1 activity ( C ) and the number of erythrocytes at the heart ( D , E ) were evaluated in 2 dpf larvae treated via bath immersion with the indicated doses of TKIs from 1 to 2 dpf. Representative images of erythrocytes are shown. The region of interest is shown. Each dot represents one individual and the mean ± SEM for each group is also shown ( C : N = 3; D , E : N is indicated in the figures). P values were calculated using one-way ANOVA and Tukey’s multiple range test ( C – E ) or log-rank test with Bonferroni correction ( B ). ns, non-significant; ** p < 0.01, *** P < 0.001 and **** P < 0.0001. ( C ) DMSO wrt: NILO p < 0.0001****, IMA p = 0.0124*, DASA p = 0.0085**, IMA p = 0.0240* or BOSU p = 0.0005***, ( D ) DMSO wrt: IMA_1 p = 0.004*** or rest of the groups p < 0.0001****, ( E ) DMSO wrt: PONA_0.1 p = 0.0062**. PONA_1 p = 0.0002*** or BOSU_1 p = 0.0374*. MFI, mean fluorescence intensity. .

Article Snippet: One dpf larvae were manually dechorionated at 24 hpf and treated for 24 h by bath immersion with the TKIs nilotinib (#HY-10159, 1 μM), imatinib (#HY-15463, 1 μM and 10 μM), dasatinib (#HY-10181, 0.1 μM and 1 μM), bosutinib (#HY-10158, 0.1 μM and 1 μM) and ponatinib (#HY-12047, 0.1 μM and 1 μM), all from MedChemExpress, diluted in egg water supplemented with 0.1% DMSO.

Techniques: Activity Assay, Fluorescence

Journal: EMBO Molecular Medicine

Article Title: TKI-mediated inhibition of NLRP1 inflammasome restores erythropoiesis in DBA syndrome

doi: 10.1038/s44321-025-00368-3

Figure Lengend Snippet: ( A , C , E ) Schematic representations of experimental designs. Zebrafish larvae were treated via bath immersion with the indicated TKIs from 1 to 2 days post-fertilization (dpf). ( B , D ) Quantification of neutrophils and representative images of treated larvae showing neutrophils (arrowheads). ( F , G ) Hemoglobin staining and quantification in Rps19-deficient zebrafish larvae generated by injecting embryos with gRNA/Cas9 complexes. Representative images of hemoglobin staining in treated larvae are shown. Regions of interest (ROI) are indicated in the images. Each dot represents one individual and the mean ± SEM for each group is also shown. P values were calculated using one-way ANOVA and Tukey’s multiple range test ( B , D , F , G ). ns, non-significant; ** p < 0.01, *** P < 0.001 and **** P < 0.0001. ( B ) DMSO wrt: DASA_01 p = 0.0162*, PONA_01 p = 0.0317*, PONA_1 p = 0.0009***, BOSU_01 p = 0.009***, BOSU_1 p = 0.0097**, ( D ) DMSO wrt: DASA_01 p = 0.0039**, DASA_1 p = 0.0317* or rest of the groups p < 0.0001****, ( F ) CONTROL wrt crRNA_rps19 p = 0.0286*, crRNA_rps19 wrt crRNA_rps19-zaka p = 0.0002***, ( G ) crRNA_STD wrt crRNA_rps19 p < 0.0001****, crRNA_rps19 wrt: Nilo p = 0.0148*, Dasa p = 0.0012**, Ima p = 0.0055**. .

Article Snippet: One dpf larvae were manually dechorionated at 24 hpf and treated for 24 h by bath immersion with the TKIs nilotinib (#HY-10159, 1 μM), imatinib (#HY-15463, 1 μM and 10 μM), dasatinib (#HY-10181, 0.1 μM and 1 μM), bosutinib (#HY-10158, 0.1 μM and 1 μM) and ponatinib (#HY-12047, 0.1 μM and 1 μM), all from MedChemExpress, diluted in egg water supplemented with 0.1% DMSO.

Techniques: Staining, Generated, Control

Journal: EMBO Molecular Medicine

Article Title: TKI-mediated inhibition of NLRP1 inflammasome restores erythropoiesis in DBA syndrome

doi: 10.1038/s44321-025-00368-3

Figure Lengend Snippet: ( A ) Chronogram of the experiment. ( B – D ) K562 cells were pretreated with 0.1 μM nilotinib ( B – E ), 0.1–1 μM imatinib and 0.1–1 μM dasatinib ( B , C ), 20–1000 nM ponatinib and 100–1000 bosutinib ( D ) for 24 h, and then differentiated with 50 μM hemin for another 24 h. Hemoglobin accumulation ( A – C ) and NLRP1, phosphorylated P38, phosphorylated JNK1/JNK2, GATA1, ZAKα and ACTB/GAPDH ( A – D ) amounts were then evaluated by Western blot. HEK293T cells were treated for 24 h with 1 µM anisomycin ( A ) in either the presence or absence of 0.1 µM nilotinib as a control of JNK1/JNK2 activation ( C ). Immunoblots are representative of three independent experiments. For ( B ) and ( D ), GATA1 and ACTB were detected on the same membrane after stripping and reprobing, whereas the remaining proteins were analyzed on separate membranes. *, GATA1. ( D ) CASP1 activity was measured by FLICA-CASP1 then analyzed by flow cytometry. Representative dot plots at different differentiation times are shown. .

Article Snippet: One dpf larvae were manually dechorionated at 24 hpf and treated for 24 h by bath immersion with the TKIs nilotinib (#HY-10159, 1 μM), imatinib (#HY-15463, 1 μM and 10 μM), dasatinib (#HY-10181, 0.1 μM and 1 μM), bosutinib (#HY-10158, 0.1 μM and 1 μM) and ponatinib (#HY-12047, 0.1 μM and 1 μM), all from MedChemExpress, diluted in egg water supplemented with 0.1% DMSO.

Techniques: Western Blot, Control, Activation Assay, Membrane, Stripping Membranes, Activity Assay, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: TKI-mediated inhibition of NLRP1 inflammasome restores erythropoiesis in DBA syndrome

doi: 10.1038/s44321-025-00368-3

Figure Lengend Snippet: ( A ) Primary human CD34 + cells were purified from human cord blood or purchased from ZenBio or StemCell Technologies, edited with CRISPR/Cas9 and differentiated for 13 days with EPO in the presence of either DMSO or 0.1 µM nilotinib. ( B – D ) Cells were stained with anti-CD235A-APC (Glycophorin A) and anti-CD71-FITC (Transferrin Receptor), or FAM FLICA, and erythroid differentiation ( B – D ) and CASP1 activity ( E ) were then analyzed by flow cytometry. Representative dot plots at different differentiation times are shown in ( B ). The differentiation score was calculated as the ratio between CD235A + /CD71 + (intermediate erythroid progenitors) and CD235A - /CD71 + (early erythroid progenitors) ( C ), and the percentage of CD235A + /CD71 high (erythroblasts) and CD235A + /CD71 low (reticulocytes) ( D ) and CASP1 activity were determined at 13 days of culture ( E ). Data are shown as the mean ± SEM ( C , E : N = 3, D : N = 2). P values were calculated using one-way ANOVA and Tukey’s multiple range test ( C , D ) or a Student’s t -test ( E ). ns, non-significant; ** p < 0.01, *** P < 0.001 and **** P < 0.0001. ( C ) gRPS19_DMSO wrt gRPS19_NILO p = 0.0420*, ( D ) Upper graph: gAAVS1_DMSO wrt: gAAVS1_NILO p = 0.0007*** or gRPS19_DMSO p = 0.0002***, gRPS19_DMSO wrt gRPS19_NILO p = 0.0001***, Lower graph: g AAVS1_DMSO wrt: gAAVS1_NILO p < 0.0001**** or gRPS19_DMSO p = 0.0123*, gRPS19_DMSO wrt gRPS19_NILO p < 0.0001***, ( E ) DMSO wrt NILO p = 0.0274*. .

Article Snippet: One dpf larvae were manually dechorionated at 24 hpf and treated for 24 h by bath immersion with the TKIs nilotinib (#HY-10159, 1 μM), imatinib (#HY-15463, 1 μM and 10 μM), dasatinib (#HY-10181, 0.1 μM and 1 μM), bosutinib (#HY-10158, 0.1 μM and 1 μM) and ponatinib (#HY-12047, 0.1 μM and 1 μM), all from MedChemExpress, diluted in egg water supplemented with 0.1% DMSO.

Techniques: Purification, CRISPR, Staining, Activity Assay, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: TKI-mediated inhibition of NLRP1 inflammasome restores erythropoiesis in DBA syndrome

doi: 10.1038/s44321-025-00368-3

Figure Lengend Snippet: ( A ) Peripheral blood mononuclear cells (PBMCs, red) and bone marrow mononuclear cells (BMMCs, gray) were isolated from 13 patients with DBAS. Cells were cultured for 2 weeks in human methylcellulose complete medium at 37 °C, with or without the indicated TKIs. Burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte macrophage (CFU-GM) colonies were counted based on standard morphological criteria. ( B – M ) Each dot represents data from an individual patient. The increase in erythroid colonies (BFU-E; B – G ) and myeloid colonies (CFU-GM; H , M ) upon treatment with TKIs is depicted. Data are shown as the mean ± SEM. ns, non-significant; ** p < 0.01, *** P < 0.001 and **** P < 0.0001 according to a Student’s t -test. ( B , C ): p < 0.0001, ( A ): p = 0.009**, ( D ): p = 0.0016**, ( E ): p = 0.0014**, ( F ): p = 0.010**, ( I ): p = 0.0023**. .

Article Snippet: One dpf larvae were manually dechorionated at 24 hpf and treated for 24 h by bath immersion with the TKIs nilotinib (#HY-10159, 1 μM), imatinib (#HY-15463, 1 μM and 10 μM), dasatinib (#HY-10181, 0.1 μM and 1 μM), bosutinib (#HY-10158, 0.1 μM and 1 μM) and ponatinib (#HY-12047, 0.1 μM and 1 μM), all from MedChemExpress, diluted in egg water supplemented with 0.1% DMSO.

Techniques: Isolation, Cell Culture