Journal: The Journal of Biological Chemistry

Article Title: Discovery of potent and selective PROTACs for the protein kinase LZK for the treatment of head and neck cancer

doi: 10.1016/j.jbc.2025.108452

Figure Lengend Snippet: Western blot analysis showing the effect of increasing concentrations of PROTACs 1 to 9 treated for 24 h on LZK expression and JNK phosphorylation in response to doxycycline (dox)-induced expression of LZK in CAL33 cells. GAPDH served as the loading control. JNK, c-Jun N-terminal kinase; LZK, leucine zipper–bearing kinase; PROTAC, proteolysis-targeting chimera.

Article Snippet: CAL33 cells (DSMZ) and SCC15 cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), 1% penicillin–streptomycin (pen–strep; Gibco), and 2 mmol/l GlutaMAX (Gibco).

Techniques: Western Blot, Expressing, Phospho-proteomics, Control

Journal: The Journal of Biological Chemistry

Article Title: Discovery of potent and selective PROTACs for the protein kinase LZK for the treatment of head and neck cancer

doi: 10.1016/j.jbc.2025.108452

Figure Lengend Snippet: Western blot analysis showing the effect of increasing concentrations of PROTACs 10 to 21 treated for 24 h on LZK expression and JNK phosphorylation in response to doxycycline (dox)-induced expression of LZK in CAL33 cells. GAPDH served as the loading control. JNK, c-Jun N-terminal kinase; LZK, leucine zipper–bearing kinase; PROTAC, proteolysis-targeting chimera.

Article Snippet: CAL33 cells (DSMZ) and SCC15 cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), 1% penicillin–streptomycin (pen–strep; Gibco), and 2 mmol/l GlutaMAX (Gibco).

Techniques: Western Blot, Expressing, Phospho-proteomics, Control

Journal: The Journal of Biological Chemistry

Article Title: Discovery of potent and selective PROTACs for the protein kinase LZK for the treatment of head and neck cancer

doi: 10.1016/j.jbc.2025.108452

Figure Lengend Snippet: Evaluation of LZK-targeting PROTAC 17 proteasome-mediated degradation ability and its effects on HNSCC cell growth . A , dose curve of the in vitro binding affinity of PROTAC 17 to LZK, assessed by Eurofin’s KdELECT KINOMEscan profiling. The amount of kinase measured by quantitative PCR (signal; y -axis) is plotted against PROTAC 17 concentration in nanometer in log10 scale ( x -axis). B , Western blot analysis showing time-course effect of PROTAC 17 on LZK expression and JNK phosphorylation in response to dox-induced LZK WT expression in CAL33 cells. GAPDH served as the loading control. C , Western blot showing dependence of PROTAC17-mediated degradation of LZK involves both ubiquitin-like molecule NEDD8 and the proteasome. LZK was induced with dox in CAL33 cells, and the cells were pretreated for 30 min with either MG132 to inhibit the proteasome or MLN4924 to inhibit NEDD8 and then exposed to the indicated concentration of PROTAC 17 for 8 h. Expression level of LZK was monitored. GAPDH served as the loading control. D and E , effect of PROTAC 17 on colony formation by indicated HNSCC cell lines. DMSO served as the vehicle control. Cells were treated for 14 days with the drug being replaced every 48 h. Plates were fixed and stained with crystal violet and visualized. Crystal violet stain was solubilized with acetic acid and measured at 595 nm. Individual data points represent technical replicates of one experiment. Data are representative of three independent experiments. Significant differences were determined by Student’s t test (∗∗∗ p < 0.001). DMSO, dimethyl sulfoxide; dox, doxycycline; HNSCC, head and neck squamous cell carcinoma; JNK, c-Jun N-terminal kinase; LZK, leucine zipper–bearing kinase; PROTAC, proteolysis-targeting chimera.

Article Snippet: CAL33 cells (DSMZ) and SCC15 cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), 1% penicillin–streptomycin (pen–strep; Gibco), and 2 mmol/l GlutaMAX (Gibco).

Techniques: In Vitro, Binding Assay, Real-time Polymerase Chain Reaction, Concentration Assay, Western Blot, Expressing, Phospho-proteomics, Control, Ubiquitin Proteomics, Staining

Journal: The Journal of Biological Chemistry

Article Title: Discovery of potent and selective PROTACs for the protein kinase LZK for the treatment of head and neck cancer

doi: 10.1016/j.jbc.2025.108452

Figure Lengend Snippet: Specificity of PROTAC 17 toward LZK . A and B , shotgun proteomic analysis of total cell protein extracts from CAL33 TR LZK WT cell line following treatment with PROTAC 17. CAL33 TR LZK WT cells were treated with doxycyline (dox) to induce LZK overexpression, followed by treatment with 500 nM PROTAC 17 or DMSO control for 24 h. LZK protein is indicated in red . Volcano plot displaying the Log2 fold change (Log2FC, x -axis) against the t test-derived −log10 statistical (n = 3) p value ( y -axis) for all proteins detected in the total cell extract from CAL33 TR LZK WT cells after dox induction of LZK overexpression. The change thresholds of Log2FC ≥|1.0| and the significance threshold of –log10 p value ≥2.0 were applied to identify proteins with levels decreased ( green boxed area ) or increased ( red boxed area ) in response to the dox treatment ( A ). Volcano plot displaying the Log2FC ( x -axis) against the t -test-derived −log10 statistical (n = 3) p value ( y -axis) for all proteins detected in the total cell extract from CAL33 TR LZK WT cells following induction of LZK overexpression and the treatment with PROTAC 17 or DMSO. The change thresholds of Log2FC ≥|1.0| and the significance threshold of –log10 p value ≥2.0 were applied to identify proteins with levels decreased ( green boxed area ) or increased ( red boxed area ) in response to the PROTAC 17 treatment ( B ). C , chemical structure of PROTAC 17 cis -epimer control. D , Western blots showing the effect of increasing concentrations of PROTAC 17 enantiomer on LZK expression and JNK phosphorylation in response to dox-induced expression of LZK in CAL33 cells. GAPDH served as the loading control. E , effect of PROTAC 17 enantiomer on colony formation by the indicated cell lines. DMSO served as the vehicle control. Cells were treated for 14 days with the drug being replaced every 48 h. Plates were fixed and stained with crystal violet and visualized. DMSO, dimethyl sulfoxide; LZK, leucine zipper–bearing kinase; PROTAC, proteolysis-targeting chimera.

Article Snippet: CAL33 cells (DSMZ) and SCC15 cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), 1% penicillin–streptomycin (pen–strep; Gibco), and 2 mmol/l GlutaMAX (Gibco).

Techniques: Over Expression, Control, Derivative Assay, Western Blot, Expressing, Phospho-proteomics, Staining