Journal: bioRxiv

Article Title: The histone demethylase KDM5 is essential for larval growth in Drosophila

doi: 10.1101/297804

Figure Lengend Snippet: (A) Lethality of kdm5 K06801 , kdm5 10424 and kdm5 140 homozygous mutant animals generated from a cross between five female and five male heterozygous parents balanced using CyO-GFP. The column labeled total flies indicates the number of progeny (adult) flies scored from at least three independent crosses. Expected number of progeny is based on Mendelian frequencies and taking into account the lethality of CyO homozygotes, i.e 33% of total adult flies. * p <0.01 (chi-squared test). (B) Position of the NP4707 , 10424 and K06801 P element insertions and molecular mapping of the kdm5 140 deletion. Ab indicates the region used to generated the rabbit polyclonal anti-KDM5 antibody ( S ecombe et al . 2007 ). (C) RT-PCR using primers to the 5’ end of the gene using RNA from whole 3 rd instar larvae. Animals homozygous for kdm5 K06801 or kdm5 10424 show low levels of transcript while kdm5 140 shows none. kdm5 mRNA normalized to wildtype ( w 1118 ) using rp49 . **** p <0.0001. (D) RT-PCR using primers to the 3’ end of the gene using RNA from whole 3 rd instar larvae. kdm5 140 has wildtype levels of the 3’ end of the transcript. **** p <0.0001. ns = not significant. (E) Western from wildtype ( w 1118 ) and kdm5 140 homozygous mutant wing imaginal discs showing KDM5 and alpha tubulin. kdm5 140 animals have no detectable full length or truncated KDM5. *ns indicates non-specific band. (F) Schematic of strain genotype for rescue of kdm5 140 with a genomic rescue transgene. Flies are homozygous for the kdm5 140 mutation on the 2 nd chromosome and homozygous for an 11kb genomic rescue transgene on the 3 rd chromosome. (G) Western blot showing KDM5 protein levels from 3 rd instar larval wing imaginal discs from wildtype ( w 1118 ) and kdm5 140 homozygotes that also have two copies of the kdm5:HA genomic rescue transgene. Anti-KDM5 (top), anti-HA (middle) and anti-histone H3 loading control (bottom). (H) kdm5 140 lethality is rescued by a transgene encoding the kdm5 locus. These data were generated by crossing female and male flies heterozygous for kdm5 140 and homozygous the wildtype genomic rescue transgene (intercross of kdm5 140 /CyO-GFP; kdm5:HA / kdm5:HA males and females).

Article Snippet: Antibodies used were anti-pH3 (Cell signaling #9701, 1/1000), anti-histone H3 (Active Motif #39763 or #39163, 1/5000), anti-alpha Tubulin (Developmental Studies Hybridoma Bank, University of Iowa; 1:5000).

Techniques: Mutagenesis, Generated, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Journal of Cancer

Article Title: Aberrant CDK9 expression within chordoma tissues and the therapeutic potential of a selective CDK9 inhibitor LDC000067.

doi: 10.7150/jca.35426

Figure Lengend Snippet: Figure 2. CDK9 expression in chordoma cell lines and tissues. (A) CDK9 expression in chordoma cell lines. (B and C) CDK9 expression in chordoma tissues. There are 2 isoforms of the CDK9 protein: the 42 KD CDK9 isoform and the 55 KD isoform. The smaller 42 KD isoform was the first identified isoform. Expression of CDK9 (55KD band) was normalized to α-Tubulin. (D) Cellular localization of CDK9 in UCH2 and CH22 cells was assessed by immunofluorescence with antibodies to CDK9 and actin.

Article Snippet: Monoclonal rabbit anti-CDK9, anti-p-RNAP II Ser-2 (13499S), antiSurvivin (2808S), anti-Bax (5023S), anti-Mcl-1(94296) and monoclonal mouse anti-α-tubulin (3874) antibodies were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Immunofluorescence

Journal: Journal of Cancer

Article Title: Aberrant CDK9 expression within chordoma tissues and the therapeutic potential of a selective CDK9 inhibitor LDC000067.

doi: 10.7150/jca.35426

Figure Lengend Snippet: Figure 3. Effect of CDK9 inhibition by siRNA on chordoma cell lines. (A and B) After transfected with increasing concentrations of CDK9 specific siRNA or nonspecific siRNA for 3 days, cell viability was determined by MTT assay after siRNA transfection in both UCH2 and CH22 cells. *p<0.05. (C and D) The morphology of UCH2 and CH22 cell lines changed after 3 days of siRNA transfection. α-Tubulin was used as a loading control. (E and F) The proteins of CDK9 and downstream proteins p-RNA II ser2 and Mcl-1 in cells were examined by Western blot after 3 days of siRNA transfection.

Article Snippet: Monoclonal rabbit anti-CDK9, anti-p-RNAP II Ser-2 (13499S), antiSurvivin (2808S), anti-Bax (5023S), anti-Mcl-1(94296) and monoclonal mouse anti-α-tubulin (3874) antibodies were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Transfection, MTT Assay, Control, Western Blot

Journal: Journal of Cancer

Article Title: Aberrant CDK9 expression within chordoma tissues and the therapeutic potential of a selective CDK9 inhibitor LDC000067.

doi: 10.7150/jca.35426

Figure Lengend Snippet: Figure 4. Effect of CDK9 inhibitor treatment on chordoma cell lines. (A and B) After exposure to increasing concentrations of CDK9 inhibitor for 120 h, cell viability was decreased in a dose-dependent manner in both UCH2 and CH22 cells, with the IC50 values for LDC000067 at 3.22 µM and 4.47 µM, respectively. (C) After incubation of UCH2 and CH22 cell lines with 1.25 µM, 2.5µM, 5.0µM, and 10.0 µM LDC000067 for 48 h, they showed a strong decrease of p-RNA II ser2 and Mcl-1 expression with increasing LDC000067 concentration. α-Tubulin was used as a loading control.

Article Snippet: Monoclonal rabbit anti-CDK9, anti-p-RNAP II Ser-2 (13499S), antiSurvivin (2808S), anti-Bax (5023S), anti-Mcl-1(94296) and monoclonal mouse anti-α-tubulin (3874) antibodies were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Incubation, Expressing, Concentration Assay, Control