pkc412  (MedChemExpress)

Journal: bioRxiv

Article Title: AXL mediates mast cell survival and resistance to tyrosine kinase inhibitors in mastocytosis

doi: 10.1101/2025.11.03.686205

Figure Lengend Snippet: (A) Proportion of cell death in parental ROSA KIT D816V cells and in cells expressing AXL WT or AXL L197M under basal conditions. (B) Immunoblot analysis of Survivin and BCL2 in ROSA KIT D816V cells expressing AXL WT, AXL L197M, or lacking AXL. Statistical significance was assessed by One-way ANOVA **p<0.01, ***p<0.001, ****p<0.0001 . (C) Growth of IL-3-dependent Ba/F3 cells expressing AXL WT or AXL L197M in presence or absence of IL-3. (D) Ingenuity pathway analysis (IPA) of RNAseq data from parental ROSA KIT D816V cells and counterparts expressing AXL WT or AXL L197M revealed clustered cellular functions. Activation z-scores were calculated by the IPA z-score algorithm, which predicts the direction of change for a function. An absolute z-score ≥ 2 is considered significant: functions are increased (pink) if z-score ≥ +2 and decreased (blue) if z-score ≤ –2. (E) Proliferation kinetics of ROSA KIT D816V cells expressing AXL WT, AXL L197M compared with control cells over 7 days of treatment with 200 nM PKC412 or DMSO. One-way ANOVA was used: ** p<0.01 : AXL -expressing cells compared to control treated with DMSO ; ## p<0.01; ### p<0.001, #### p<0.0001 : AXL -expressing cells compared to control cells treated with PKC412 ). (F) Cell death measured by SYTOX blue staining after 96-hour treatment with 200 nM PKC412 or DMSO. Significance was determined relative to controls and DMSO by two-way ANOVA (***p<0.001, ****p<0.0001 ). (G) Immunoblot analysis of BCL-XL, BCL2, MCL1, and cleaved caspase-3/procaspase-3 ratio in ROSA KIT D816V cells expressing AXL WT, AXL L197M, or control, following 48-hour treatment with 200 nM PKC412 or DMSO. Statistical significance was assessed versus DMSO-treated cells using two-way ANOVA (*p<0.05; **p<0.01). Data are representative of ≥3 independent experiments, except cleaved caspase-3/procaspase-3 (n=2).

Article Snippet: Following 48 hours of treatment with PKC412 (200 nM), or DMSO, expression levels of BCL-XL (sc-56021, Santa cruz), BCL-2 (M0887, Dako), MCL1 (ab32087, Abcam), and caspase-3 (sc-7272, Santa cruz) were assessed.

Techniques: Expressing, Western Blot, Activation Assay, Control, Staining

Journal: bioRxiv

Article Title: AXL mediates mast cell survival and resistance to tyrosine kinase inhibitors in mastocytosis

doi: 10.1101/2025.11.03.686205

Figure Lengend Snippet: (A) Cell viability of ROSA KIT D816V cells (control, AXL WT, or AXL L197M) measured by WST-8 assay after7 days of treatment with DMSO, 200 nM PKC412, 500 nM R428 or their combination. Viability was determined by absorbance at 450 nm. (B) Representative images of cell confluence at day7. (C) Immunoblots analysis of BCL2, BCL-XL, MCL1, Caspase 3 performed in cells after 48 h of treatments as in A. β-actin or HSP70 served for normalization. (D) Expression of Livin and cIPA1 assessed by immunoblots in cells treated for 48 hours, as indicated. Data are representative of three independent experiments. (E) Fresh PBMCs from patient MCL3 were treated with DMSO, 400 nM PKC412, 1 µM R428, or their combination. MCs viability (CD117⁺, FcεRI⁺, Sytox Blue⁻) was monitored by FACS over 21-days. Due to limited cell availability, only days 6 and 14 were analyzed in duplicate. Statistical significance was assessed by one-way ANOVA (A, D, E). Caspase analyses were performed by two-way ANOVA (C). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Following 48 hours of treatment with PKC412 (200 nM), or DMSO, expression levels of BCL-XL (sc-56021, Santa cruz), BCL-2 (M0887, Dako), MCL1 (ab32087, Abcam), and caspase-3 (sc-7272, Santa cruz) were assessed.

Techniques: Control, Western Blot, Expressing