Journal: Cancer Research Communications

Article Title: The Lysine Demethylase KDM4C Is an Oncogenic Driver and Regulates ERK Activity in KRAS-Mutant Pancreatic Ductal Adenocarcinoma

doi: 10.1158/2767-9764.CRC-25-0278

Figure Lengend Snippet: KDM4C is differentially upregulated in pancreatic cancer and correlates with poor prognosis. A, Western blots show human PDAC cell lines overexpress KDM4C compared with the noncancerous pancreatic epithelial cell line HPNE. B, Representative photomicrographs of KDM4C expression by IHC on TMAs of archival PDAC samples demonstrates upregulation of KDM4C in neoplastic cells compared with matched nonneoplastic pancreatic tissues. C, Bar graph showing the mean and distribution of the immunoscores of KDM4C expression in 187 cases of PDAC and matched normal pancreas. The score difference between the groups was assessed for statistical significance via paired t test; ****, P ≤ 0.0001. D, Kaplan–Meier survival plot showing increased survival of patients with low-KDM4C expressing PDAC ( n = 96) compared with high-expressing tumors ( n = 90) from the TMA dataset ( P = 0.03). To determine KDM4C expression, we used PDAC TMAs, comprised of 187 PDAC cases. From each case, two punches (1.5 mm) from separate tumor areas (T1 and T2) and one punch (1.5 mm) from the normal pancreas (N) were included. After IHC staining with anti-KDM4C antibody, the samples were evaluated by a pathologist for both the intensity and percentage of stained cells in a semi-quantitative way. Intensity: 0: no staining/1: weak/2: moderate/3: strong. Proportion: 1: less than 10%/2: 10% to 50%/3: 50% to 75%/4: more than 75%. The final immunoscore was calculated by adding the intensity score to the proportion score according to the Allred method. The average immunoscore score from the tumor area was divided by the immunoscore from the matched normal area for each case. The log 2 ratio was then used for survival analysis, with a threshold log 2 ratio of 0.32.

Article Snippet: Primary antibody incubations were carried out overnight at 4°C using the following antibodies: rat anti–E-cadherin (1:100, Thermo Fisher Scientific, cat. #14-3249-82, RRID: AB_1210458), rabbit anti-pERK (1:100, Cell Signaling Technology, cat. #9101, RRID: AB_331646), rabbit anti-KDM4C (1:100, Novus, cat. #NBP1-49600, RRID: AB_10011699), mouse anti-H3K9Me3 (1:100, Active Motif, cat. #61013, RRID: AB_2687870), and mouse anti-HA tag (1:100, Thermo Fisher Scientific, cat. #26183-A488, RRID: AB_2610624).

Techniques: Western Blot, Expressing, Immunohistochemistry, Staining

Journal: Cancer Research Communications

Article Title: The Lysine Demethylase KDM4C Is an Oncogenic Driver and Regulates ERK Activity in KRAS-Mutant Pancreatic Ductal Adenocarcinoma

doi: 10.1158/2767-9764.CRC-25-0278

Figure Lengend Snippet: KDM4C is differentially upregulated in pancreatic cancer and correlates with poor prognosis. A, Western blots show human PDAC cell lines overexpress KDM4C compared with the noncancerous pancreatic epithelial cell line HPNE. B, Representative photomicrographs of KDM4C expression by IHC on TMAs of archival PDAC samples demonstrates upregulation of KDM4C in neoplastic cells compared with matched nonneoplastic pancreatic tissues. C, Bar graph showing the mean and distribution of the immunoscores of KDM4C expression in 187 cases of PDAC and matched normal pancreas. The score difference between the groups was assessed for statistical significance via paired t test; ****, P ≤ 0.0001. D, Kaplan–Meier survival plot showing increased survival of patients with low-KDM4C expressing PDAC ( n = 96) compared with high-expressing tumors ( n = 90) from the TMA dataset ( P = 0.03). To determine KDM4C expression, we used PDAC TMAs, comprised of 187 PDAC cases. From each case, two punches (1.5 mm) from separate tumor areas (T1 and T2) and one punch (1.5 mm) from the normal pancreas (N) were included. After IHC staining with anti-KDM4C antibody, the samples were evaluated by a pathologist for both the intensity and percentage of stained cells in a semi-quantitative way. Intensity: 0: no staining/1: weak/2: moderate/3: strong. Proportion: 1: less than 10%/2: 10% to 50%/3: 50% to 75%/4: more than 75%. The final immunoscore was calculated by adding the intensity score to the proportion score according to the Allred method. The average immunoscore score from the tumor area was divided by the immunoscore from the matched normal area for each case. The log 2 ratio was then used for survival analysis, with a threshold log 2 ratio of 0.32.

Article Snippet: Then 1 mg of total protein in 500 μL volume was incubated with 1 μg respective antibodies: anti-SIRT1 (1:100, Abcam, cat. #ab110304, RRID: AB_10864359), anti-PARP1 (1:100, Cell Signaling Technology, cat. #9542, RRID: AB_2160739), anti-ZNF148 (1:100, Sigma-Aldrich, cat. #HPA001656, RRID: AB_1079995), anti-KDM4C (1:100, Novus, cat. #NBP1-49600, RRID: AB_10011699), or anti-IgG control (1:100, Thermo Fisher Scientific, cat. #14-4714-82, RRID: AB_470111), overnight at 4°C, with rotation, whereas 25 μg of lysate was reserved as input control.

Techniques: Western Blot, Expressing, Immunohistochemistry, Staining

Journal: Cancer Research Communications

Article Title: The Lysine Demethylase KDM4C Is an Oncogenic Driver and Regulates ERK Activity in KRAS-Mutant Pancreatic Ductal Adenocarcinoma

doi: 10.1158/2767-9764.CRC-25-0278

Figure Lengend Snippet: KDM4C is differentially upregulated in pancreatic cancer and correlates with poor prognosis. A, Western blots show human PDAC cell lines overexpress KDM4C compared with the noncancerous pancreatic epithelial cell line HPNE. B, Representative photomicrographs of KDM4C expression by IHC on TMAs of archival PDAC samples demonstrates upregulation of KDM4C in neoplastic cells compared with matched nonneoplastic pancreatic tissues. C, Bar graph showing the mean and distribution of the immunoscores of KDM4C expression in 187 cases of PDAC and matched normal pancreas. The score difference between the groups was assessed for statistical significance via paired t test; ****, P ≤ 0.0001. D, Kaplan–Meier survival plot showing increased survival of patients with low-KDM4C expressing PDAC ( n = 96) compared with high-expressing tumors ( n = 90) from the TMA dataset ( P = 0.03). To determine KDM4C expression, we used PDAC TMAs, comprised of 187 PDAC cases. From each case, two punches (1.5 mm) from separate tumor areas (T1 and T2) and one punch (1.5 mm) from the normal pancreas (N) were included. After IHC staining with anti-KDM4C antibody, the samples were evaluated by a pathologist for both the intensity and percentage of stained cells in a semi-quantitative way. Intensity: 0: no staining/1: weak/2: moderate/3: strong. Proportion: 1: less than 10%/2: 10% to 50%/3: 50% to 75%/4: more than 75%. The final immunoscore was calculated by adding the intensity score to the proportion score according to the Allred method. The average immunoscore score from the tumor area was divided by the immunoscore from the matched normal area for each case. The log 2 ratio was then used for survival analysis, with a threshold log 2 ratio of 0.32.

Article Snippet: After 1 hour of blocking in 5% milk (RRID: ABT), membranes were incubated with either anti-KDM4C (1:500 dilution; Santa Cruz Biotechnology, cat. #sc-515767, RRID: AB_3068588), anti–pERK1/2 (1:1,000; Cell Signaling Technology, cat. #9101, RRID: AB_331646), anti–ERK1/2 (1:1,000; Cell Signaling Technology, cat. #9102, RRID: AB_330744), anti-KDM4A (1:1,000; Cell Signaling Technology, cat. #5328, RRID: AB_10828595), anti-KDM4B (1:1,000; Cell Signaling Technology, cat. #8639, RRID: AB_11140642), anti-KDM4D (1:1,000; Santa Cruz Biotechnology, cat. #sc-393750, RRID: AB_3674229), anti-KDM4E (1:1,000, Thermo Fisher Scientific, cat. #PA5-71128, RRID: AB_2691349), anti-DUSP2 (1:300, Santa Cruz Biotechnology, cat. #sc-32776, RRID: AB_2094883), or horseradish peroxidase (HRP)-conjugated anti–β-actin (1:10,000; Santa Cruz Biotechnology, cat. #sc-47778, RRID: AB_626632) primary antibodies at 4°C overnight.

Techniques: Western Blot, Expressing, Immunohistochemistry, Staining