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Journal: The Journal of Biological Chemistry

Article Title: Lysine demethylase KDM1A promotes cell growth via FKBP8–BCL2 axis in hepatocellular carcinoma

doi: 10.1016/j.jbc.2022.102374

Figure Lengend Snippet: Cytoplasmic KDM1A promotes HCC cell growth . A and B , left panel , same number of HLF control or KDM1A-KD cells were seeded. Cell proliferation over 5 days was measured with CCK-8. Fold of growth was normalized to that in control cells. Right panel shows Western blot (WB) for indicated cells. Error bars denote standard deviation of six biological replicates. p Value was calculated by one-way ANOVA followed by pair-wise comparison as indicated. C , 500 HLF control or KDM1A-KD cells were seeded in 6-well plate. About 14 days later, cell colonies were stained with crystal violet . D and E , HLF control cells ( D ) or cells expressing Myc-KDM1A ( E ) were fractionated into cytoplasm (Cyto) and nucleus (Nuc). Shown are the WB results with indicated antibodies. F , GFP-KDM1A were expressed in HLF cells with lentivirus. Shown are photos taken with fluorescent microscopy. The scale bar represents 20 μm. G and H , left panel , same number of HLF KDM1A-KD and rescue cells were seeded. Cell proliferation over 5 days was measured with CCK-8. Fold of growth was normalized to that in control cells. Right panel shows WB for indicated cells. (Ctrl for control, KD for KDM1A-KD, WT for KDM1A-WT rescue, AA for K114A/R115A rescue, AA-DN for K114A/R115A–A539E/K661A rescue). Error bars denote standard deviation of six biological replicates. p Value was calculated by one-way ANOVA. CCK-8, Cell Counting Kit-8; HCC, hepatocellular carcinoma; KD, knockdown; KDM1A, lysine demethylase 1A.

Article Snippet: Coexpression with KDM1A-WT but not inactive mutant decreased FKBP8 methylation ( E ).Consistently, treatment with KDM1A selective inhibitor ORY1001 (TargetMol; catalog no.: T6922) increased FKBP8 methylation ( F ).

Techniques: Control, CCK-8 Assay, Western Blot, Standard Deviation, Comparison, Staining, Expressing, Microscopy, Cell Counting, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Lysine demethylase KDM1A promotes cell growth via FKBP8–BCL2 axis in hepatocellular carcinoma

doi: 10.1016/j.jbc.2022.102374

Figure Lengend Snippet: KDM1A–K117 is acetylated by KAT8. A , FLAG-KDM1A was cotransfected with HA-tagged acetyltransferases into 293T cells. KDM1A acetylation was analyzed with IP–WB. B , FLAG-KDM1A was cotransfected with increasing HA-KAT8 into 293T cells. KDM1A acetylation was analyzed with IP–WB. C , FLAG-KDM1A was cotransfected with HA-KAT8-WT or inactive -K274A (DN) into 293T cells. KDM1A acetylation was analyzed with IP–WB. D , FLAG-KDM1A fragments were cotransfected with HA-KAT8 into 293T cells. FLAG-KDM1A acetylation was analyzed with IP–WB. E , FLAG-KDM1A-WT or -K117R was cotransfected with KAT8 into 293T cells. KDM1A acetylation was analyzed with IP–WB. F , FLAG-KDM1A-WT or -K117R was cotransfected with KAT8 into 293T cells. Cells were then treated with 3.3 μM TSA and/or 20 mM NIM for 4 h before collection. KDM1A acetylation was analyzed with IP–WB. HA, hemagglutinin; IP, immunoprecipitation; K117, lysine 117; KAT8, lysine acetyltransferase 8; KDM1A, lysine demethylase 1A; NIM, nicotinamide; TSA, trichostatin A; WB, Western blot.

Article Snippet: Coexpression with KDM1A-WT but not inactive mutant decreased FKBP8 methylation ( E ).Consistently, treatment with KDM1A selective inhibitor ORY1001 (TargetMol; catalog no.: T6922) increased FKBP8 methylation ( F ).

Techniques: Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Lysine demethylase KDM1A promotes cell growth via FKBP8–BCL2 axis in hepatocellular carcinoma

doi: 10.1016/j.jbc.2022.102374

Figure Lengend Snippet: K117 acetylation promotes cytoplasmic localization and protein stability of KDM1A. A and B , KDM1A-WT or mutants were rescue expressed in HLF KDM1A-KD cells. A , shown is the result of immunofluorescence with myc-tag antibody. The scale bar represents 20 μm. B , cells were fractionated into cytoplasmic and nuclear fractions. Fractions were analyzed with WB. C , KDM1A-WT or mutants were expressed in HLF cells, whereas endogenous KDM1A was knocked down with doxycycline-inducible shRNA. Cells were analyzed with WB. D , 0.2 μg Myc-KDM1A-WT or mutants were transiently cotransfected with 0.05 μg GFP into 293T cells in 3.5 cm dish. Cells were analyzed with WB with GFP and actin as loading control. E , KAT8 was knocked down in HLF cells with lentivirus-expressed shRNA. Cells were analyzed with WB. F and G , 0.4 μg Myc-KDM1A-WT or 0.2 μg -K117Q, -K114A/R115A was transfected into 293T cells in 3.5 cm dish. About 24 h later, cells were split equally into three or four dishes as indicated. The next day, cells were treated with 25 μg/ml Chx for indicated time and analyzed with WB. H , 0.4 μg KDM1A-WT or 0.2 μg KDM1A-K114A/K115A were transfected into control or JADE2-KD 293T cells in 3.5 cm dish. About 24 h later, cells were split equally into three dishes as indicated. The next day, cells were then treated with 25 μg/ml Chx for indicated time and analyzed with WB. Chx, cycloheximide; JADE2, Jade family PHD finger 2; K117, lysine 117; KD, knockdown; KDM1A, lysine demethylase 1A; WB, Western blot.

Article Snippet: Coexpression with KDM1A-WT but not inactive mutant decreased FKBP8 methylation ( E ).Consistently, treatment with KDM1A selective inhibitor ORY1001 (TargetMol; catalog no.: T6922) increased FKBP8 methylation ( F ).

Techniques: Immunofluorescence, shRNA, Control, Transfection, Knockdown, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Lysine demethylase KDM1A promotes cell growth via FKBP8–BCL2 axis in hepatocellular carcinoma

doi: 10.1016/j.jbc.2022.102374

Figure Lengend Snippet: KDM1A–FKBP8–BCL2 axis increases cellular resistance to sorafenib. A , 293T cells were transfected with GST-FKBP8, Myc-SMYD3, Myc-KDM1A, and HA-KAT8. Cells were analyzed with GST-pulldown (PD) and WB. B and C , KDM1A-WT or KDM1A–K117R was rescue expressed in HLF KDM1A-KD cells. Cells were analyzed with WB ( B ). In ( C ), cells were treated with 5 μM sorafenib for 80 h. Cell proliferation was measured with CCK-8. Fold of cell proliferation was normalized to that of control cells. Error bars denote standard deviation of six biological replicates. p Value was calculated with one-way ANOVA followed by pair-wise comparison as indicated. D , WB analysis for sorafenib-resistant HLF cell. E , KDM1A was knocked down in sorafenib-resistant HLF cells. Cells were treated with 5 μM sorafenib for 4 days. Cell proliferation was measured with CCK-8. Fold of cell proliferation was normalized to that of control. Error bars denote standard deviation of six biological replicates. p Value was calculated with one-way ANOVA followed by pair-wise comparison as indicated. F , primary HCC cell culture 1 was treated with 10 μM GSK2879552 (GSK) or 10 μM ORY1001 (ORY) for 5 days. Cells were analyzed with WB. G , primary HCC cell culture 1 was infected with lentivirus expressing KAT8-shRNA. After selection with puromycin for 5 days, cell lysates were analyzed with WB. H , primary HCC cell culture 1 was treated with 5 μM sorafenib and/or 10 μM ORY1001. Cell proliferation was measured with CCK-8. Fold of cell proliferation was normalized to that of control. Error bars denote standard deviation of six biological replicates. p Value was calculated with one-way ANOVA followed by pair-wise comparison as indicated. BCL2, B-cell lymphoma-2; CCK-8, Cell Counting Kit-8; FKBP8, FKBP prolyl isomerase 8; GST, glutathione- S -transferase; HA, hemagglutinin; HCC, hepatocellular carcinoma; KAT8, lysine acetyltransferase 8; KDM1A, lysine demethylase 1A; SMYD3, SET and MYND domain–containing 3; WB, Western blot.

Article Snippet: Coexpression with KDM1A-WT but not inactive mutant decreased FKBP8 methylation ( E ).Consistently, treatment with KDM1A selective inhibitor ORY1001 (TargetMol; catalog no.: T6922) increased FKBP8 methylation ( F ).

Techniques: Transfection, CCK-8 Assay, Control, Standard Deviation, Comparison, Cell Culture, Infection, Expressing, shRNA, Selection, Cell Counting, Western Blot

Journal: Cancers

Article Title: LncRNA LINC00518 Acts as an Oncogene in Uveal Melanoma by Regulating an RNA-Based Network

doi: 10.3390/cancers12123867

Figure Lengend Snippet: Identification of TFs potentially regulating LINC00518 expression. ( A ) Experimentally validated transcription factor binding sites (TFBSs) upstream of the transcription start site of LINC00518 locus. ( B ) Expression analysis after KDM1A inhibition achieved by ORY-1001. ( C , D ) Expression analysis after MITF inhibition through A-485 treatment in 92.1 and MEL270 cells, respectively. Correlation of expression with MITF is shown. Each experiment was performed in three in vitro replicates. *: p -value < 0.05; **: p -value < 0.005; ***: p -value < 0.0005.

Article Snippet: KDM1A activity was inhibited by treating UM cell lines with ORY-1001 (TargetMol, Boston, MA, USA), while inhibition of MITF was achieved by A-485 (MedChemExpress, Princeton, NJ, USA), which induces a reduction in both mRNA and protein levels of MITF [ ].

Techniques: Expressing, Binding Assay, Inhibition, In Vitro