Journal: bioRxiv

Article Title: KDM4A promotes NEPC progression through regulation of MYC expression

doi: 10.1101/2022.05.14.491739

Figure Lengend Snippet: ( A ) Venn Diagram showing KDM4A and KDM5D as the two histone lysine demethylases that are overexpressed in NEPC using the Beltran et al. RNA-seq dataset. ( B ) The expression of KDM4A and KDM5D in NEPC compared to adeno-CRPC and SCPC. ( C ) Kdm4a mRNA is upregulated in prostate tumors from older PNR mice than tumors from young PNR mice, PN mice and PN het R het mice. ( D ) KDM4A IHC in primary NEPC and adenocarcinoma-CRPC. ( E ) KDM4A IHC staining in prostate cancer PDXs. ( F ) KDM4A IHC in primary tumors from PS, PTR, PSTR and TRAMP mice. ( G ) Correlation of KDM4A mRNA expression with NE markers CHGA and CHGB in the Beltran et al. RNA-seq dataset.

Article Snippet: Primary antibodies used for immunohistochemistry are as follows: KDM4A (ab191433, Abcam), KDM4A (3393S, CST), Ki-67 (GTX16667, GeneTex), Synaptophysin (M7315, Dako), AR (ab133273, Abcam), Chromogranin A (20086, ImmunoStar).

Techniques: RNA Sequencing, Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: KDM4A promotes NEPC progression through regulation of MYC expression

doi: 10.1101/2022.05.14.491739

Figure Lengend Snippet: ( A-B ) Kdm4a KD in PSTR cells, as confirmed by Western blot (A), led to significant reduced growth in foci-forming assay in vitro (B). ( C-D ). Kdm4a KD in PTR cells, as confirmed by Western blot (C), led to significant reduced growth in vitro (D). ( E-F ) Kdm4a KO in PTR cells, as confirmed by Western blot (E), led to significant reduced growth in foci-forming assay in vitro (F). ( G-H ) KDM4A KD in 144-13 cells, as confirmed by Western blot (G), led to significant reduced cell number (H). ( I-J ) KDM4A KD in 144-13 cells (I) or Kdm4a KO in PSTR cells (J) led to reduced number of colonies and reduced colony sizes using soft agar colony formation assay.

Article Snippet: Primary antibodies used for immunohistochemistry are as follows: KDM4A (ab191433, Abcam), KDM4A (3393S, CST), Ki-67 (GTX16667, GeneTex), Synaptophysin (M7315, Dako), AR (ab133273, Abcam), Chromogranin A (20086, ImmunoStar).

Techniques: Western Blot, In Vitro, Soft Agar Assay

Journal: bioRxiv

Article Title: KDM4A promotes NEPC progression through regulation of MYC expression

doi: 10.1101/2022.05.14.491739

Figure Lengend Snippet: ( A ) Kdm4a KO delayed the growth of PSTR cells in subQ implantation model. ( B ) IHC staining confirmed the loss of KDM4A expression in tumors from KT compound mutant mice compared to tumors from TRAMP mice. ( C ) Reduced tumor weights in KT mice (20-28 weeks) compared to tumors from age-matched TRAMP mice. ( D ) H & E staining of prostate tumors from 20-week-old TRAMP and KT mice. ( E ) Kaplan Meier survival analysis comparing KT mice to TRAMP mice. ( F-G ) Ki67 IHC staining in prostate tumors from TRAMP and KT mice. ( H ) The incidences of prostate adenocarcinoma and NEPC in TRAMP and KT mice. ( I ) IHC staining of SYP in prostate tumors from >30-week-old and <30-week-old TRAMP mice and KT mice.

Article Snippet: Primary antibodies used for immunohistochemistry are as follows: KDM4A (ab191433, Abcam), KDM4A (3393S, CST), Ki-67 (GTX16667, GeneTex), Synaptophysin (M7315, Dako), AR (ab133273, Abcam), Chromogranin A (20086, ImmunoStar).

Techniques: Immunohistochemistry, Expressing, Mutagenesis, Staining

Journal: bioRxiv

Article Title: KDM4A promotes NEPC progression through regulation of MYC expression

doi: 10.1101/2022.05.14.491739

Figure Lengend Snippet: ( A ) GSEA analysis of RNA-seq on Kdm4a -KD and control PTR cells showed that MYC signaling is suppressed in Kdm4a -KD cells. ( B ) Fold changes in the expression of selective MYC-target genes comparing Kdm4a -KD cells to control cells. ( C-D ) GSEA analysis of Beltran et al. RNA-seq data (C) and Ku et al. RNA-seq data showed that MYC signaling is activated in NEPC compared to prostate adenocarcinoma. ( E-F ) Western blot showed that KDM4A KD led to reduced MYC expression in PSTR (E) and PTR (F) cells. ( G ) qRT-PCR showed that Kdm4a KD led to reduced Myc mRNA expression in PTR cells. ( H ) ChIP-seq analyses identified MYC as a KDM4A-target gene in 144-13 cells. ( I ) MYC KD led to reduced cell proliferation in 144-13 cells. ( J ) MYC inhibitor MYCi975 led to reduced cell proliferation in 144-13 cells.

Article Snippet: Primary antibodies used for immunohistochemistry are as follows: KDM4A (ab191433, Abcam), KDM4A (3393S, CST), Ki-67 (GTX16667, GeneTex), Synaptophysin (M7315, Dako), AR (ab133273, Abcam), Chromogranin A (20086, ImmunoStar).

Techniques: RNA Sequencing, Control, Expressing, Western Blot, Quantitative RT-PCR, ChIP-sequencing

Journal: bioRxiv

Article Title: KDM4A promotes NEPC progression through regulation of MYC expression

doi: 10.1101/2022.05.14.491739

Figure Lengend Snippet: ( A ) KDM4 inhibitor QC6352 treatment led to increased total H3K9me3 and H3K36me3 in PSTR cells. ( B ) QC6352 suppressed PSTR growth in vitro as shown by foci-forming assay. ( C-D ) QC6352 treatment induced apoapsis in PSTR cells as shown by Western blot analysis of cleaved caspase 3 and cleaved PARP1 and fluorescence imaging of caspase 3/7 substrates. ( E ) QC6352 suppressed the growth of PTR cells. ( F ) QC6352 induced apoptosis in PTR cells as shown by WB analysis of cleaved caspase 3 and cleaved PARP1. ( G ) QC6352 and NCGC00244536 suppressed the growth of 144-13 cells in vitro . ( H ) Kdm4a -KO blunt the effect of QC6352 treatment on cell growth in vitro .

Article Snippet: Primary antibodies used for immunohistochemistry are as follows: KDM4A (ab191433, Abcam), KDM4A (3393S, CST), Ki-67 (GTX16667, GeneTex), Synaptophysin (M7315, Dako), AR (ab133273, Abcam), Chromogranin A (20086, ImmunoStar).

Techniques: In Vitro, Western Blot, Fluorescence, Imaging

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) Actively growing U2OS cells (confluency 60–70%) were fixed following extraction with 1% Triton X-100 and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (Ab1 Bethyl Lab, panel 1) and human Pol-I largest subunit A194 (panel 2); nuclear DNA was stained by DAPI (panel 3). Images were acquired with a Leica fluorescent microscope (DM5000- 20 × objective). Representative nuclei are shown with associated merged images (panel 4). Scale bar, 5 μM. ( b ) A diagram of the human rDNA repeat is shown indicating the positions of eight sets of specific PCR primer/probes used for qPCR analysis of immunoprecipitated DNA. 5′ETS, 5′-external transcribed spacer; IGS, intergenic spacer; Prom, the rRNA promoter, term, the terminator. Signal representing the transcribed region (TrR) is the average of the combined signal from 5′ETS, 18S, 5.8S and 28S rRNA. Signal representing the non-transcribed region (nTrR) is the average of the combined signal from IGS1 and IGS2. ( c ) ChIP assays were performed using antibodies specific to human KDM4A from two different sources and analysed by qPCR using eight sets of specific probes and primers derived from different regions of rDNA repeats (see the diagram above). The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) expressed as % from total chromatin input (see for raw data). Signal representing the transcribed region (TrR) is the average of the combined signal from 5′ETS, 18S, 5.8S and 28S rRNA. Signal representing the non-transcribed region (nTrR) is the average of the combined signal from IGS1 and IGS2. The s.d.'s from three independent experiments are shown; n =3. ( d , e ) KDM4A was immunoprecipitated from nuclear extract of U2OS cells using KMD4A-specific antibodies (Bethyl). Immunoprecipitated complexes were analysed by western blotting using either antibodies specific to human Pol-I largest subunit A194 and UBF (lane 4), or to SL1 subunits TAF 1 110 and TAF 1 63 (lane 7). Input—lanes 1, 2 and 6, negative control (anti-HA antibodies)—lanes 3 and 8. Purified SL1—lane 5. Positions of prestained molecular weight markers (PageRuler Plus, Fermentas) are indicated.

Article Snippet: Cells were then incubated at 4 °C overnight with the indicated primary antibodies: Rabbit polyclonal anti-JMJD2A (Bethyl A-300-861-A; 0.5 μg ml −1 ), Rabbit polyclonal anti-KDM4A antibodies (Sigma, HPA007610, 0.5 μg ml −1 ), Rabbit polyclonal anti-HA (Cell Signaling C29F4, 1 μg ml −1 ), RPA 194 Santa Cruz SC-48385 1 μg ml −1 , mouse monoclonal anti-UBF (SC-13125 1 μg ml −1 ), mouse monoclonal anti-BrdU 2 μg ml −1 (BU-33, Sigma), mouse monoclonal anti-myc antibodies (9E10, Santa Cruz).

Techniques: Immunofluorescence, Staining, Microscopy, Immunoprecipitation, Derivative Assay, Negative Control, Western Blot, Purification, Molecular Weight

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) Schematic representation of the labelling of cells with 3 H-uridine to determine the effect of KDM4A depletion on ongoing rRNA synthesis. RNA was extracted 2 and 4 h after 3 H-uridine addition and de nov o rRNA transcripts were detected by tritium imaging of RNA blots as in . ( b ) The relative efficiencies of rRNA synthesis were quantitated as in and transcript levels are indicated for 47S/45S pre-rRNA. The data, expressed as a percentage of the highest value (set at 100%). The s.d.'s for three independent experiments are shown; n =3. ( c ) The relative efficiencies of cell growth were determined using MTT assay. The s.d.'s for four independent samples are shown; n =4. ( d ) Growing U2OS cells (∼70% confluent) were electroporated with an expression vector encoding either wild-type KDM4A (WT) or catalytically inactive mutant (MUT). Cells were grown for 48 h and then 5-FUrd was added for 30 min and cells were fixed and analysed by indirect immunofluorescence using antibodies specific to 5-FUrd (anti-BrdU antibodies); nuclear DNA was stained by DAPI. ( e ) Box-and-whiskers plot of the quantification of 5-FUrd staining per cell ( n =100–200) obtained from the experiment shown in d using R open source software (a.u. stands for arbitrary units). Note that the 5-FUrd levels of the cell populations overexpressing the MUT and the WT are not significantly different ( P value=0.637, Wilcoxon test). The median values are shown as horizontal lines, outliers are shown as open circles; n =3.

Article Snippet: Cells were then incubated at 4 °C overnight with the indicated primary antibodies: Rabbit polyclonal anti-JMJD2A (Bethyl A-300-861-A; 0.5 μg ml −1 ), Rabbit polyclonal anti-KDM4A antibodies (Sigma, HPA007610, 0.5 μg ml −1 ), Rabbit polyclonal anti-HA (Cell Signaling C29F4, 1 μg ml −1 ), RPA 194 Santa Cruz SC-48385 1 μg ml −1 , mouse monoclonal anti-UBF (SC-13125 1 μg ml −1 ), mouse monoclonal anti-BrdU 2 μg ml −1 (BU-33, Sigma), mouse monoclonal anti-myc antibodies (9E10, Santa Cruz).

Techniques: Imaging, MTT Assay, Expressing, Plasmid Preparation, Mutagenesis, Immunofluorescence, Staining, Software

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) Schematic representation of the labelling of cells with 3 H-uridine to determine the effect of KDM4A depletion on activation of rRNA synthesis. U2OS cells were transfected either with non-targeting (siScr) or KDM4A-specific siRNA3 (siA3). 24 h post-transfection cells were starved. Transcription was activated by addition of serum and 3 H-uridine at time point 0. ( b ) RNA was extracted 15, 30, 60 and 90 min after serum addition and de nov o rRNA transcripts were detected by tritium imaging of RNA blots (top panel) using X-ray film. Total 18S and 28S rRNAs were detected by ethidium bromide staining (bottom panel). ( c ) To determine the relative efficiencies of rRNA synthesis, RNA blots were imaged using tritium image plate (Fuji) and quantitated with aid of phosphoimager (Fuji) and Aida software (Raytec). Transcript levels are indicated for 47S/45S pre-rRNA. The data are expressed as a percentage of the highest value (set at 100%). The s.d.'s for three independent experiments are shown; n =3. ( d ). Schematic representation of the labelling of cells with 5-FUrd to determine the effect of KDM4A depletion on activation of rRNA synthesis in individual cells. U2OS cells were serum-starved for 16 h, electroporated with either a non-targeting siRNA (siScr) or two siRNAs directed against KDM4A (siA1 and siA2) and kept in serum-free medium for further 24 h before serum refeeding in the presence of 5-FUrd for 30 min. ( e ). Cells were stained for 5-FUrd incorporation using anti-BrdU antibodies. Nuclear DNA was stained with DAPI. Representative immunofluorescence images are shown. Scale bar, 5 μM. ( f ). Quantification of 5-FUrd staining shown in e , done as in ; P value (Wilcoxon test)= * 4.84 × 10 −11 , ** 6.12 × 10 −12 . Note the presence of many outliers in samples transfected by KDM4A siRNA, which most likely correspond to untransfected cells. The median values are shown as horizontal lines, outliers are shown as open circles; n =3.

Article Snippet: Cells were then incubated at 4 °C overnight with the indicated primary antibodies: Rabbit polyclonal anti-JMJD2A (Bethyl A-300-861-A; 0.5 μg ml −1 ), Rabbit polyclonal anti-KDM4A antibodies (Sigma, HPA007610, 0.5 μg ml −1 ), Rabbit polyclonal anti-HA (Cell Signaling C29F4, 1 μg ml −1 ), RPA 194 Santa Cruz SC-48385 1 μg ml −1 , mouse monoclonal anti-UBF (SC-13125 1 μg ml −1 ), mouse monoclonal anti-BrdU 2 μg ml −1 (BU-33, Sigma), mouse monoclonal anti-myc antibodies (9E10, Santa Cruz).

Techniques: Activation Assay, Transfection, Imaging, Staining, Software, Immunofluorescence

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ). U2OS cells were serum starved for 24 h ( t =0) and refed with serum. Cells were fixed at 30 and 60 min following serum addition, and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (Ab1 Bethyl Lab, panels 1) and human Pol-I largest subunit A194 (panels 2); nuclear DNA was stained by DAPI (panels 3). Merged images are shown (panel 4). Scale bar, 5 μM. ( b ) ChIP assays were performed using antibodies specific to human KDM4A (Ab1, Bethyl Lab) and chromatin isolated from starved and starved–refed cells (30 and 60 min after serum addition) and analysed as in to determine KDM4A binding to rDNA promoter (Pr), transcribed region (TrR) or non-transcribed region (nTrR). The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) expressed as % from total chromatin input (see for raw data). Standard deviations from three independent experiments are shown; n =3. ( c ) ChIP assays were performed using antibodies specific to human KDM4A as in b and chromatin immunoprecipitated from starved and starved–refed cells (60 min after serum addition) were analysed by qPCR for the presence either CDC6 or P0 or GAPDH promoters representing a positive and negative controls of the anti-KDM4A ChIP, respectively. The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) expressed as % from total chromatin input (see for raw data). Standard deviations from three independent experiments are shown; n =3.

Article Snippet: Cells were then incubated at 4 °C overnight with the indicated primary antibodies: Rabbit polyclonal anti-JMJD2A (Bethyl A-300-861-A; 0.5 μg ml −1 ), Rabbit polyclonal anti-KDM4A antibodies (Sigma, HPA007610, 0.5 μg ml −1 ), Rabbit polyclonal anti-HA (Cell Signaling C29F4, 1 μg ml −1 ), RPA 194 Santa Cruz SC-48385 1 μg ml −1 , mouse monoclonal anti-UBF (SC-13125 1 μg ml −1 ), mouse monoclonal anti-BrdU 2 μg ml −1 (BU-33, Sigma), mouse monoclonal anti-myc antibodies (9E10, Santa Cruz).

Techniques: Immunofluorescence, Staining, Isolation, Binding Assay, Negative Control, Immunoprecipitation

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) U2OS cells were electroporated with an expression vector encoding either wild-type HA-tagged KDM4A (WT) or catalytically inactive mutant (MUT) or with empty vector (vector) together with a siRNA targeting the 5′UTR of KDM4A, to knockdown endogenous KDM4A while allowing expression of exogenous KDM4A proteins. Cells were grown for 24 h and then starved for another 24 h before serum refeeding in the presence of 5-FUrd. 30 min after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to HA-tag to detect overexpressed proteins, (panels 1), 5-FUrd (panels 2); nuclear DNA was stained by DAPI (panels 3). Merged images are shown (panels 4). Bar=5 μM. ( b ) Quantification of 5-FUrd staining shown in a , done as in . Note that the 5-FUrd levels of the cell populations overexpressing the MUT and the WT are significantly different ( P value=4.97 × 10 −9 , Wilcoxon test). The median values are shown as horizontal lines, outliers are shown as open circles; n =3.

Article Snippet: Cells were then incubated at 4 °C overnight with the indicated primary antibodies: Rabbit polyclonal anti-JMJD2A (Bethyl A-300-861-A; 0.5 μg ml −1 ), Rabbit polyclonal anti-KDM4A antibodies (Sigma, HPA007610, 0.5 μg ml −1 ), Rabbit polyclonal anti-HA (Cell Signaling C29F4, 1 μg ml −1 ), RPA 194 Santa Cruz SC-48385 1 μg ml −1 , mouse monoclonal anti-UBF (SC-13125 1 μg ml −1 ), mouse monoclonal anti-BrdU 2 μg ml −1 (BU-33, Sigma), mouse monoclonal anti-myc antibodies (9E10, Santa Cruz).

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) Chromatin was isolated from starved and starved–refed cells (30 min after serum addition) and subjected for the first round of immunoprecipitation using antibody specific to Pol-I subunit A135. After elution chromatin was subjected to the second IP round using antibody specific to KDM4A and analysed by qPCR as in . The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) expressed as % from total chromatin input (see for raw data). Standard deviations from three independent experiments are shown; n =3. ( b , c ) Chromatin was isolated from starved ( b ) and starved–refed U2OS cells (1 h after serum addition) ( c ) and the same quantities of a chromatin were subjected for the first round of immunoprecipitation using antibody specific to Pol-I subunit A135. After elution, the chromatin was subjected for the second IP round using antibodies specific to histone H3, H3K9me1, H3K9me2 and H3K9me3 and analysed by qPCR as in . The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) normalized to the specific H3 signal (representing the difference between the signals from H3 specific antibody and an appropriate IgG, see for raw data) and expressed as % from total chromatin input. Standard deviations from three independent experiments are shown (see for raw data); n =3. ( d , e ). U2OS cells were transfected with KDM4A specific siRNA3. 24 h post-transfection cells were starved for 24 h. Chromatin was isolated from starved ( d ) and starved–refed cells (1 h after serum addition) ( e ). Chromatin was analysed as in b and c (see for raw data); n =3.

Article Snippet: Cells were then incubated at 4 °C overnight with the indicated primary antibodies: Rabbit polyclonal anti-JMJD2A (Bethyl A-300-861-A; 0.5 μg ml −1 ), Rabbit polyclonal anti-KDM4A antibodies (Sigma, HPA007610, 0.5 μg ml −1 ), Rabbit polyclonal anti-HA (Cell Signaling C29F4, 1 μg ml −1 ), RPA 194 Santa Cruz SC-48385 1 μg ml −1 , mouse monoclonal anti-UBF (SC-13125 1 μg ml −1 ), mouse monoclonal anti-BrdU 2 μg ml −1 (BU-33, Sigma), mouse monoclonal anti-myc antibodies (9E10, Santa Cruz).

Techniques: Isolation, Immunoprecipitation, Negative Control, Transfection

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) U2OS cells were serum starved for 24 h. Starved cells were incubated for 30 min with MAPK inhibitor PD98059 (PD), mTOR inhibitor rapamycin (Rapa), or PI3K inhibitor LY294002 (LY) or a mixture of mTOR and MAPK inhibitors (PD+Rapa) or DMSO as a control (Untr) and refed with serum in the presence of 5-FUrd and the respective inhibitors. Thirty minutes after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (Ab1, panels 1) and 5-FUrd (panels 2); nuclear DNA was stained by DAPI (panels 3). Scale bar, 5 μM. ( b ) U2OS cells were serum starved for 24 h. Starved cells were incubated for 30 min with PI3K inhibitor PI-103, AKT inhibitor AKTVIII (AKT-i), or DMSO (Untr) and refed with serum in the presence of 5-FUrd and the respective inhibitors. Thirty minutes after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (Ab1, panels 1) and 5-FUrd (panels 2); nuclear DNA was stained by DAPI (panels 3). ( c ) Quantification of 5-FUrd staining shown in a and b done as in . The effect of inhibitors or combination of inhibitors are all significant ( P value<10 4 , Wilcoxon test), except treatment with PD alone. Note that no incorporation could be measured upon AKT inhibition. The median values are shown as horizontal lines, outliers are shown as open circles; n =3. ( d ) U2OS cells were transfected with an expression vector encoding c-myc-tagged PTEN either wild-type (PTEN-WT) or mutated for its lipid phosphatase activity (PTEN-m). 24 h following transfection, cells were serum starved for 24 h and refed with serum containing 5-FUrd. Cells were fixed and analysed by indirect immunofluorescence using antibodies specific to c-myc Tag to detect overexpressed PTEN (panels 1); KDM4A (panels 2); nuclear DNA was stained by DAPI (panels 3).

Article Snippet: Cells were then incubated at 4 °C overnight with the indicated primary antibodies: Rabbit polyclonal anti-JMJD2A (Bethyl A-300-861-A; 0.5 μg ml −1 ), Rabbit polyclonal anti-KDM4A antibodies (Sigma, HPA007610, 0.5 μg ml −1 ), Rabbit polyclonal anti-HA (Cell Signaling C29F4, 1 μg ml −1 ), RPA 194 Santa Cruz SC-48385 1 μg ml −1 , mouse monoclonal anti-UBF (SC-13125 1 μg ml −1 ), mouse monoclonal anti-BrdU 2 μg ml −1 (BU-33, Sigma), mouse monoclonal anti-myc antibodies (9E10, Santa Cruz).

Techniques: Incubation, Immunofluorescence, Staining, Inhibition, Transfection, Expressing, Plasmid Preparation, Activity Assay

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) U2OS cells were serum starved for 24 h. Starved cells were incubated for 30 min with PDK1 inhibitor GSK2334470 (PDK1i), or PI3K inhibitor LY294002 (LY) or DMSO as a control (Untr) and refed with serum in the presence of 5-FUrd and the respective inhibitors (+serum) or left starved (no serum). Thirty minutes after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (panels 1) and Pol-I (panels 2); nuclear DNA was stained by DAPI (panels 3). Scale bar, 5 μM. ( b ) U2OS cells were serum starved for 24 h. Starved cells were incubated for 30 min with SGK1 inhibitor GSK650394 (SGK1i), or DMSO as a control (Untr) and refed with serum in the presence of 5-FUrd and inhibitor (+serum) or left starved (no serum). Thirty minutes after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (panels 1) and Pol-I (panels 2); nuclear DNA was stained by DAPI (panels 3). Scale bar, 5 μM. ( c ) U2OS cells were electroporated with either a non-targeting siRNA (siScr) or two siRNAs directed against SGK1 (siSGK1-1 and siSGK1-2) and kept in serum-free medium for further 24 h before serum refeeding in the presence of 5-FUrd. Thirty minutes after serum addition, cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (panels 1) and Pol-I (panels 2); nuclear DNA was stained by DAPI (panels 3). Scale bar, 5 μM.

Article Snippet: Cells were then incubated at 4 °C overnight with the indicated primary antibodies: Rabbit polyclonal anti-JMJD2A (Bethyl A-300-861-A; 0.5 μg ml −1 ), Rabbit polyclonal anti-KDM4A antibodies (Sigma, HPA007610, 0.5 μg ml −1 ), Rabbit polyclonal anti-HA (Cell Signaling C29F4, 1 μg ml −1 ), RPA 194 Santa Cruz SC-48385 1 μg ml −1 , mouse monoclonal anti-UBF (SC-13125 1 μg ml −1 ), mouse monoclonal anti-BrdU 2 μg ml −1 (BU-33, Sigma), mouse monoclonal anti-myc antibodies (9E10, Santa Cruz).

Techniques: Incubation, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: Effect of the inhibition of various serum-activated kinases on the activation of rDNA transcription and on the nucleolar localization of KDM4A.

Article Snippet: Cells were then incubated at 4 °C overnight with the indicated primary antibodies: Rabbit polyclonal anti-JMJD2A (Bethyl A-300-861-A; 0.5 μg ml −1 ), Rabbit polyclonal anti-KDM4A antibodies (Sigma, HPA007610, 0.5 μg ml −1 ), Rabbit polyclonal anti-HA (Cell Signaling C29F4, 1 μg ml −1 ), RPA 194 Santa Cruz SC-48385 1 μg ml −1 , mouse monoclonal anti-UBF (SC-13125 1 μg ml −1 ), mouse monoclonal anti-BrdU 2 μg ml −1 (BU-33, Sigma), mouse monoclonal anti-myc antibodies (9E10, Santa Cruz).

Techniques: Inhibition, Activation Assay, Over Expression

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: PI3K–Akt, mTOR and MAPK pathways regulate rRNA gene transcription by targeting RNA Polymerase I machinery (as previously published, see Discussion). PI3K–PDK1–SGK1 signalling cascade regulates activation of rRNA gene transcription by controlling histone modifications at rDNA loci through KDM4A localization. SGK1 may in addition target Pol-I transcription machinery independently of KDM4A.

Article Snippet: Cells were then incubated at 4 °C overnight with the indicated primary antibodies: Rabbit polyclonal anti-JMJD2A (Bethyl A-300-861-A; 0.5 μg ml −1 ), Rabbit polyclonal anti-KDM4A antibodies (Sigma, HPA007610, 0.5 μg ml −1 ), Rabbit polyclonal anti-HA (Cell Signaling C29F4, 1 μg ml −1 ), RPA 194 Santa Cruz SC-48385 1 μg ml −1 , mouse monoclonal anti-UBF (SC-13125 1 μg ml −1 ), mouse monoclonal anti-BrdU 2 μg ml −1 (BU-33, Sigma), mouse monoclonal anti-myc antibodies (9E10, Santa Cruz).

Techniques: Activation Assay