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Localization and signaling pathway adaption after restoration of FN1 expression in imatinib-resistant cells. (A,B) Immunofluorescence staining of imatinib-resistant K-562 cells transfected with the negative control (NC) or a FN1 -encoding plasmid (p FN1 ). FN1 was stained using Alexa Fluor 488 (green) with co-staining of vimentin, ZO-1 or LAMP1 using Alexa Fluor 594 (red) and DAPI (blue). Depicted is one representative picture for each staining of N = 3 as merge picture. 63× magnification. Bar = 10 µm. Arrows indicate cytosolic, membrane and lysosomal staining. (B) FN1 co-localization with vimentin, ZO-1 or LAMP1 after densitometric analysis of N = 3 given in %. Error bars indicate standard deviation. (C,D) Recurrent genome-wide gene expression changes in TKI resistance. (C) STRING interaction network obtained for 33 recurrently deregulated genes in imatinib- and nilotinib-resistant cell lines compared to treatment-naïve cells. Pink: experimentally determined, green: gene neighborhood, yellow: textmining, black: co-expression. Red arrow indicates number of upregulated genes; green number of downregulated genes. (D) mRNA expression of COL15A1 and <t>SPARC</t> analyzed by RT-qPCR. Expression was investigated in imatinib-resistant cells without BCR::ABL1 mutations (IM-R1, IM-R2, WT), imatinib-resistant cells harboring BCR::ABL1 p. E255K (E255K), as well as nilotinib-resistant cell (N-R1, N-R2) compared to treatment-naïve K-562 cells. Data were normalized to GAPDH and TBP and treatment-naïve cells. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. N = 3. Error bars indicate standard deviation. *** p < 0.001. (E) Phosphorylation of p-38 after restoration of FN1 expression in nilotinib-resistant cells. HSP90 is shown as a housekeeping protein. Depicted is one representative blot out of N = 3. P-p38, phosho-p-38.
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Localization and signaling pathway adaption after restoration of FN1 expression in imatinib-resistant cells. (A,B) Immunofluorescence staining of imatinib-resistant K-562 cells transfected with the negative control (NC) or a FN1 -encoding plasmid (p FN1 ). FN1 was stained using Alexa Fluor 488 (green) with co-staining of vimentin, ZO-1 or LAMP1 using Alexa Fluor 594 (red) and DAPI (blue). Depicted is one representative picture for each staining of N = 3 as merge picture. 63× magnification. Bar = 10 µm. Arrows indicate cytosolic, membrane and lysosomal staining. (B) FN1 co-localization with vimentin, ZO-1 or LAMP1 after densitometric analysis of N = 3 given in %. Error bars indicate standard deviation. (C,D) Recurrent genome-wide gene expression changes in TKI resistance. (C) STRING interaction network obtained for 33 recurrently deregulated genes in imatinib- and nilotinib-resistant cell lines compared to treatment-naïve cells. Pink: experimentally determined, green: gene neighborhood, yellow: textmining, black: co-expression. Red arrow indicates number of upregulated genes; green number of downregulated genes. (D) mRNA expression of COL15A1 and <t>SPARC</t> analyzed by RT-qPCR. Expression was investigated in imatinib-resistant cells without BCR::ABL1 mutations (IM-R1, IM-R2, WT), imatinib-resistant cells harboring BCR::ABL1 p. E255K (E255K), as well as nilotinib-resistant cell (N-R1, N-R2) compared to treatment-naïve K-562 cells. Data were normalized to GAPDH and TBP and treatment-naïve cells. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. N = 3. Error bars indicate standard deviation. *** p < 0.001. (E) Phosphorylation of p-38 after restoration of FN1 expression in nilotinib-resistant cells. HSP90 is shown as a housekeeping protein. Depicted is one representative blot out of N = 3. P-p38, phosho-p-38.
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Localization and signaling pathway adaption after restoration of FN1 expression in imatinib-resistant cells. (A,B) Immunofluorescence staining of imatinib-resistant K-562 cells transfected with the negative control (NC) or a FN1 -encoding plasmid (p FN1 ). FN1 was stained using Alexa Fluor 488 (green) with co-staining of vimentin, ZO-1 or LAMP1 using Alexa Fluor 594 (red) and DAPI (blue). Depicted is one representative picture for each staining of N = 3 as merge picture. 63× magnification. Bar = 10 µm. Arrows indicate cytosolic, membrane and lysosomal staining. (B) FN1 co-localization with vimentin, ZO-1 or LAMP1 after densitometric analysis of N = 3 given in %. Error bars indicate standard deviation. (C,D) Recurrent genome-wide gene expression changes in TKI resistance. (C) STRING interaction network obtained for 33 recurrently deregulated genes in imatinib- and nilotinib-resistant cell lines compared to treatment-naïve cells. Pink: experimentally determined, green: gene neighborhood, yellow: textmining, black: co-expression. Red arrow indicates number of upregulated genes; green number of downregulated genes. (D) mRNA expression of COL15A1 and <t>SPARC</t> analyzed by RT-qPCR. Expression was investigated in imatinib-resistant cells without BCR::ABL1 mutations (IM-R1, IM-R2, WT), imatinib-resistant cells harboring BCR::ABL1 p. E255K (E255K), as well as nilotinib-resistant cell (N-R1, N-R2) compared to treatment-naïve K-562 cells. Data were normalized to GAPDH and TBP and treatment-naïve cells. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. N = 3. Error bars indicate standard deviation. *** p < 0.001. (E) Phosphorylation of p-38 after restoration of FN1 expression in nilotinib-resistant cells. HSP90 is shown as a housekeeping protein. Depicted is one representative blot out of N = 3. P-p38, phosho-p-38.
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Localization and signaling pathway adaption after restoration of FN1 expression in imatinib-resistant cells. (A,B) Immunofluorescence staining of imatinib-resistant K-562 cells transfected with the negative control (NC) or a FN1 -encoding plasmid (p FN1 ). FN1 was stained using Alexa Fluor 488 (green) with co-staining of vimentin, ZO-1 or LAMP1 using Alexa Fluor 594 (red) and DAPI (blue). Depicted is one representative picture for each staining of N = 3 as merge picture. 63× magnification. Bar = 10 µm. Arrows indicate cytosolic, membrane and lysosomal staining. (B) FN1 co-localization with vimentin, ZO-1 or LAMP1 after densitometric analysis of N = 3 given in %. Error bars indicate standard deviation. (C,D) Recurrent genome-wide gene expression changes in TKI resistance. (C) STRING interaction network obtained for 33 recurrently deregulated genes in imatinib- and nilotinib-resistant cell lines compared to treatment-naïve cells. Pink: experimentally determined, green: gene neighborhood, yellow: textmining, black: co-expression. Red arrow indicates number of upregulated genes; green number of downregulated genes. (D) mRNA expression of COL15A1 and <t>SPARC</t> analyzed by RT-qPCR. Expression was investigated in imatinib-resistant cells without BCR::ABL1 mutations (IM-R1, IM-R2, WT), imatinib-resistant cells harboring BCR::ABL1 p. E255K (E255K), as well as nilotinib-resistant cell (N-R1, N-R2) compared to treatment-naïve K-562 cells. Data were normalized to GAPDH and TBP and treatment-naïve cells. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. N = 3. Error bars indicate standard deviation. *** p < 0.001. (E) Phosphorylation of p-38 after restoration of FN1 expression in nilotinib-resistant cells. HSP90 is shown as a housekeeping protein. Depicted is one representative blot out of N = 3. P-p38, phosho-p-38.
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Localization and signaling pathway adaption after restoration of FN1 expression in imatinib-resistant cells. (A,B) Immunofluorescence staining of imatinib-resistant K-562 cells transfected with the negative control (NC) or a FN1 -encoding plasmid (p FN1 ). FN1 was stained using Alexa Fluor 488 (green) with co-staining of vimentin, ZO-1 or LAMP1 using Alexa Fluor 594 (red) and DAPI (blue). Depicted is one representative picture for each staining of N = 3 as merge picture. 63× magnification. Bar = 10 µm. Arrows indicate cytosolic, membrane and lysosomal staining. (B) FN1 co-localization with vimentin, ZO-1 or LAMP1 after densitometric analysis of N = 3 given in %. Error bars indicate standard deviation. (C,D) Recurrent genome-wide gene expression changes in TKI resistance. (C) STRING interaction network obtained for 33 recurrently deregulated genes in imatinib- and nilotinib-resistant cell lines compared to treatment-naïve cells. Pink: experimentally determined, green: gene neighborhood, yellow: textmining, black: co-expression. Red arrow indicates number of upregulated genes; green number of downregulated genes. (D) mRNA expression of COL15A1 and <t>SPARC</t> analyzed by RT-qPCR. Expression was investigated in imatinib-resistant cells without BCR::ABL1 mutations (IM-R1, IM-R2, WT), imatinib-resistant cells harboring BCR::ABL1 p. E255K (E255K), as well as nilotinib-resistant cell (N-R1, N-R2) compared to treatment-naïve K-562 cells. Data were normalized to GAPDH and TBP and treatment-naïve cells. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. N = 3. Error bars indicate standard deviation. *** p < 0.001. (E) Phosphorylation of p-38 after restoration of FN1 expression in nilotinib-resistant cells. HSP90 is shown as a housekeeping protein. Depicted is one representative blot out of N = 3. P-p38, phosho-p-38.
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Localization and signaling pathway adaption after restoration of FN1 expression in imatinib-resistant cells. (A,B) Immunofluorescence staining of imatinib-resistant K-562 cells transfected with the negative control (NC) or a FN1 -encoding plasmid (p FN1 ). FN1 was stained using Alexa Fluor 488 (green) with co-staining of vimentin, ZO-1 or LAMP1 using Alexa Fluor 594 (red) and DAPI (blue). Depicted is one representative picture for each staining of N = 3 as merge picture. 63× magnification. Bar = 10 µm. Arrows indicate cytosolic, membrane and lysosomal staining. (B) FN1 co-localization with vimentin, ZO-1 or LAMP1 after densitometric analysis of N = 3 given in %. Error bars indicate standard deviation. (C,D) Recurrent genome-wide gene expression changes in TKI resistance. (C) STRING interaction network obtained for 33 recurrently deregulated genes in imatinib- and nilotinib-resistant cell lines compared to treatment-naïve cells. Pink: experimentally determined, green: gene neighborhood, yellow: textmining, black: co-expression. Red arrow indicates number of upregulated genes; green number of downregulated genes. (D) mRNA expression of COL15A1 and <t>SPARC</t> analyzed by RT-qPCR. Expression was investigated in imatinib-resistant cells without BCR::ABL1 mutations (IM-R1, IM-R2, WT), imatinib-resistant cells harboring BCR::ABL1 p. E255K (E255K), as well as nilotinib-resistant cell (N-R1, N-R2) compared to treatment-naïve K-562 cells. Data were normalized to GAPDH and TBP and treatment-naïve cells. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. N = 3. Error bars indicate standard deviation. *** p < 0.001. (E) Phosphorylation of p-38 after restoration of FN1 expression in nilotinib-resistant cells. HSP90 is shown as a housekeeping protein. Depicted is one representative blot out of N = 3. P-p38, phosho-p-38.
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Localization and signaling pathway adaption after restoration of FN1 expression in imatinib-resistant cells. (A,B) Immunofluorescence staining of imatinib-resistant K-562 cells transfected with the negative control (NC) or a FN1 -encoding plasmid (p FN1 ). FN1 was stained using Alexa Fluor 488 (green) with co-staining of vimentin, ZO-1 or LAMP1 using Alexa Fluor 594 (red) and DAPI (blue). Depicted is one representative picture for each staining of N = 3 as merge picture. 63× magnification. Bar = 10 µm. Arrows indicate cytosolic, membrane and lysosomal staining. (B) FN1 co-localization with vimentin, ZO-1 or LAMP1 after densitometric analysis of N = 3 given in %. Error bars indicate standard deviation. (C,D) Recurrent genome-wide gene expression changes in TKI resistance. (C) STRING interaction network obtained for 33 recurrently deregulated genes in imatinib- and nilotinib-resistant cell lines compared to treatment-naïve cells. Pink: experimentally determined, green: gene neighborhood, yellow: textmining, black: co-expression. Red arrow indicates number of upregulated genes; green number of downregulated genes. (D) mRNA expression of COL15A1 and SPARC analyzed by RT-qPCR. Expression was investigated in imatinib-resistant cells without BCR::ABL1 mutations (IM-R1, IM-R2, WT), imatinib-resistant cells harboring BCR::ABL1 p. E255K (E255K), as well as nilotinib-resistant cell (N-R1, N-R2) compared to treatment-naïve K-562 cells. Data were normalized to GAPDH and TBP and treatment-naïve cells. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. N = 3. Error bars indicate standard deviation. *** p < 0.001. (E) Phosphorylation of p-38 after restoration of FN1 expression in nilotinib-resistant cells. HSP90 is shown as a housekeeping protein. Depicted is one representative blot out of N = 3. P-p38, phosho-p-38.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Association of fibronectin 1 deregulation with tyrosine kinase inhibitor resistance in chronic myeloid leukemia

doi: 10.3389/fcell.2025.1725857

Figure Lengend Snippet: Localization and signaling pathway adaption after restoration of FN1 expression in imatinib-resistant cells. (A,B) Immunofluorescence staining of imatinib-resistant K-562 cells transfected with the negative control (NC) or a FN1 -encoding plasmid (p FN1 ). FN1 was stained using Alexa Fluor 488 (green) with co-staining of vimentin, ZO-1 or LAMP1 using Alexa Fluor 594 (red) and DAPI (blue). Depicted is one representative picture for each staining of N = 3 as merge picture. 63× magnification. Bar = 10 µm. Arrows indicate cytosolic, membrane and lysosomal staining. (B) FN1 co-localization with vimentin, ZO-1 or LAMP1 after densitometric analysis of N = 3 given in %. Error bars indicate standard deviation. (C,D) Recurrent genome-wide gene expression changes in TKI resistance. (C) STRING interaction network obtained for 33 recurrently deregulated genes in imatinib- and nilotinib-resistant cell lines compared to treatment-naïve cells. Pink: experimentally determined, green: gene neighborhood, yellow: textmining, black: co-expression. Red arrow indicates number of upregulated genes; green number of downregulated genes. (D) mRNA expression of COL15A1 and SPARC analyzed by RT-qPCR. Expression was investigated in imatinib-resistant cells without BCR::ABL1 mutations (IM-R1, IM-R2, WT), imatinib-resistant cells harboring BCR::ABL1 p. E255K (E255K), as well as nilotinib-resistant cell (N-R1, N-R2) compared to treatment-naïve K-562 cells. Data were normalized to GAPDH and TBP and treatment-naïve cells. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. N = 3. Error bars indicate standard deviation. *** p < 0.001. (E) Phosphorylation of p-38 after restoration of FN1 expression in nilotinib-resistant cells. HSP90 is shown as a housekeeping protein. Depicted is one representative blot out of N = 3. P-p38, phosho-p-38.

Article Snippet: RT-qPCR was performed using TaqMan Universal Master Mix II, with UNG (Thermo Fisher Scientific) and the assays FN1 (Hs01549976_m1), SPARC (Hs00234160_m1), COL15A1 (Hs00266332_m1), GAPDH (Hs02786624_g1), TBP (Hs00427620_m1) and 18S (Hs99999901_s1) with default cycling conditions.

Techniques: Expressing, Immunofluorescence, Staining, Transfection, Negative Control, Plasmid Preparation, Membrane, Standard Deviation, Genome Wide, Gene Expression, Quantitative RT-PCR, Phospho-proteomics