trc kinase shrna library  (Thermo Fisher)

Journal: iScience

Article Title: Selective regulation of chemosensitivity in glioblastoma by phosphatidylinositol 3-kinase beta

doi: 10.1016/j.isci.2024.109921

Figure Lengend Snippet: PIK3CB/PI3Kβ, but not other PI3K kinases, activates PI3K signaling and regulates TMZ sensitivity in MGMT-deficient GBMs (A) Combinations of TMZ and knockdown of individual PI3K kinases. PI3Kβ-high SF295 or PI3Kβ-low LN229 cells were transfected with viruses harboring non-silencing (NS) shRNA or shRNA of PIK3CA, PIK3CB, or PIK3CD. Cells were then treated with DMSO or 200 μM TMZ. 4 days after treatment, cell viability was measured using the MTS assay. Cells treated with shNS and DMSO serve as controls (100% of viability). (B) Knockout of PI3Kα or PI3Kβ. PI3Kα/β-high U87MG cells were transduced with viruses harboring gRNA of non-targeting (NT) or PIK3CA. PI3Kβ-high SF295 cells were transduced with viruses harboring gRNA of NT or PIK3CB. Levels of PI3Kα, PI3Kβ, or PI3K signaling were monitored using immunoblotting. β-actin (ACTB) was the loading control. (C) Combinations of TMZ and knockout of PI3Kα or PI3Kβ. PI3Kα/β-high U87MG or PI3Kβ-high SF295 cells were transduced with viruses having gRNAs of NT, PIK3CA, or PIK3CB. Cells were then treated with DMSO or 200 μM TMZ. 4 days after treatments, cell viability was measured using the MTS viability assay. Cells treated with NT gRNA and DMSO serve as controls (100% of viability). (D) Overexpression of active AKT1, AKT2, or AKT3. PI3Kα/β-high U87MG cells were transduced with viruses having pBABE (vector control) or plasmids with active myristoylated AKT isoforms (pBABE-Myr-AKT1, pBABE-Myr-AKT2, or pBABE-Myr-AKT3). Cells were then treated with 20 μM of TGX-221 (PI3Kβ-selective inhibitor) and 200 μM TMZ. Cell viability was measured using the MTS assay. Cells treated with DMSO and pBABE serve as controls (100% of viability). Error bars are standard deviations derived from three to four independent replicates. Student’s t test and One-Way ANOVA were used to determine p values. ns: not significant.

Article Snippet: We previously purchased a TRC kinase shRNA library from Thermo Fisher Scientific, which was discontinued recently.

Techniques: Knockdown, Transfection, shRNA, MTS Assay, Knock-Out, Transduction, Western Blot, Control, Viability Assay, Over Expression, Plasmid Preparation, Derivative Assay

Journal: iScience

Article Title: Selective regulation of chemosensitivity in glioblastoma by phosphatidylinositol 3-kinase beta

doi: 10.1016/j.isci.2024.109921

Figure Lengend Snippet:

Article Snippet: We previously purchased a TRC kinase shRNA library from Thermo Fisher Scientific, which was discontinued recently.

Techniques: Recombinant, Transfection, Proliferation Assay, Concentration Assay, Software, Protein Array, Expressing, shRNA, Modification

Journal: Epigenetics

Article Title: An shRNA kinase screen identifies regulators of UHRF1 stability and activity in mouse embryonic stem cells

doi: 10.1080/15592294.2022.2044126

Figure Lengend Snippet: High-throughput shRNA screen-identified kinases mediating loss of UHRF1 in Serum/LIF to 2i transition in mESCs. a) Loss of UHRF1 in the transition to ground state pluripotency is mediated by the proteasome. ESCs grown in 2i conditions for a total of 10 days were subsequently cultured in the presence of the 26S proteasome inhibitor MG132 at a final concentration of 10 μM for a total of 6 hours. Western blot analysis was performed for 2x replicates of Serum/LIF, 2i+DMSO and 2i+MG132 ESCs. Note full length UHRF1 has 2 bands indicated by arrows with the slower migrating band considered to be a modified form of UHRF1. b) MG132 does not impact UHRF1 at the transcriptional level. Data are presented as 2− ΔΔCt with Gapdh serving as a loading control and the relative expression levels of Uhrf1 in 2i and 2i+MG132 ESCs are normalized to the level of expression in Serum/LIF ESCs. Error bars represent the standard error of the mean from 3 replicates. c) Schematic detailing the experimental design for the screen, asterisks denotes samples that were collected for subsequent sequencing – Serum/LIF day 0, Serum/LIF day 7, 2i day 7 GFP+ and 2i day 7GFP-. Cells which are GFP positive still express UHRF1 despite being gown in 2i conditions, enrichment of shRNA hairpins in this population of cells indicate that the factor targeted potentially plays a role in UHRF1 degradation upon resetting in 2i conditions. The experiment was performed with 3 replicates. d) MDS plot showing clustering of samples, the plot shows that 2i GFP+ samples (black circles) cluster separately from Serum/LIF day 0 (red), Serum/LIF day 7 (green) and 2i GFP- samples (dark blue). e) Plot showing the fold change (on the logarithmic scale) of each hairpin (GFP+ versus GFP-) along with the average count per million of the hairpin in the GFP+ and GFP- populations (on the logarithmic scale). The dashed blue lines represent a 2-fold increase and 2-fold decrease within the GFP+population. Hairpins enriched within the GFP+, and therefore potentially involved in the regulation of URHF1, are highlighted in the red circle. Significant candidates circled are listed in Supplementary Table 1.

Article Snippet: To perform the screen we obtained the TRC lentiviral mouse kinase shRNA library from the Broad Institute.

Techniques: High Throughput Screening Assay, shRNA, Cell Culture, Concentration Assay, Western Blot, Modification, Control, Expressing, Sequencing

Journal: Epigenetics

Article Title: An shRNA kinase screen identifies regulators of UHRF1 stability and activity in mouse embryonic stem cells

doi: 10.1080/15592294.2022.2044126

Figure Lengend Snippet: Knockdown of Nags rescues expression of UHRF1 in 2i conditions. a) Nags KD ESCs grown in 2i conditions display an altered heterogeneous morphology, data is shown for Serum/LIF KD and 2i Nags KD ESCs generated by shRNA4. b) Western blot analysis for UHRF1 upon knockdown of Nags. Analysis was performed for cell lines generated from the single shRNA producing a successful knockdown (shRNA4) and additionally shRNA1 that failed to knockdown Nags when measured by qPCR. The Western blot was performed in duplicate along with a positive control (Serum/LIF ESCs), as well as cells transduced with a non-silencing control vector grown in 2i conditions (NS 2i). Densitometry analysis was performed displaying the density of the UHRF1 band relative to the GAPDH band, error bars represent the minimum and maximum values from 2 technical replicates. The results show that knockdown of Nags prevents degradation of UHRF1 in cells cultured in 2i conditions, and therefore replicates the results of the shRNA screen. Furthermore, shRNA1 failed to impact upon UHRF1 expression. c) Six candidate genes whose expression is known to change from Serum/LIF to 2i were measured in Nags KD ESCs shRNA4, grown in both Serum/LIF and 2i conditions. Data are presented as 2− ΔCt with Gapdh serving as a loading control. Error bars represent the standard error of the mean from 3 technical replicates, * denotes P value < 0.05. d) Expression of the pluripotency marker NANOG as measured by immunofluorescence. Data is shown for Nags KD ESCs grown in both Serum/LIF and 2i. Data is shown for 2 representative images, and DAPI was used as a nuclear stain. e) DNA methylation in each KD cell line is shown in the two panels. Data is shown as average of all CpG values in each amplicon (as detailed in Supplementary Figure 6 and methods section) and error bars represent the standard deviation.

Article Snippet: To perform the screen we obtained the TRC lentiviral mouse kinase shRNA library from the Broad Institute.

Techniques: Knockdown, Expressing, Generated, Western Blot, shRNA, Positive Control, Transduction, Control, Plasmid Preparation, Cell Culture, Marker, Immunofluorescence, Staining, DNA Methylation Assay, Amplification, Standard Deviation

Journal: Epigenetics

Article Title: An shRNA kinase screen identifies regulators of UHRF1 stability and activity in mouse embryonic stem cells

doi: 10.1080/15592294.2022.2044126

Figure Lengend Snippet: Knockdown of Sephs2 rescues expression of UHRF1 in 2i conditions. a) Sephs2 KD ESCs grown in Serum/LIF and 2i conditions display an altered morphology, data is shown for Serum/LIF and 2i Sephs2 KD ESCs generated by shRNA3. b) Western blot analysis for UHRF1 upon knockdown of Sephs2. Analysis was performed for cell lines generated from the two shRNAs producing a successful knockdown. The Western blot was performed in duplicate along with a positive control (Serum/LIF ESCs), as well as cells transduced with a non-silencing control vector grown in 2i conditions (NS 2i). Densitometry analysis was performed displaying the density of the UHRF1 band relative to the GAPDH band, error bars represent the minimum and maximum values from 2 technical replicates. The results show that knock-down of Sephs2 prevents degradation of UHRF1 in cells cultured in 2i conditions, and therefore replicates the results of the shRNA screen. c) Six candidate genes whose expression is known to change in Serum/LIF to 2i were measured in Sephs2 KD ESCs grown in both Serum/LIF and 2i conditions. Data is shown for Serum/LIF Sephs2 KD ESCs (Serum/LIF shRNA3), and 2i ESCs transduced with the 2 effective shRNA downregulating Sephs2 (shRNA2 and shRNA3). Data are presented as 2− ΔCt with Gapdh serving as a loading control. For the Serum/LIF Sephs2 KD ESCs data is shown for ESCs transduced with shRNA 3 targeting Sephs2. Error bars represent the standard error of the mean from 3 technical replicates, * denotes P value < 0.05. d) Expression of the pluripotency marker NANOG as measured by immunofluorescence. Data is shown for Sephs2 KD ESCs grown in both Serum/LIF and 2i conditions similar to . e) DNA methylation in each KD cell line is shown in the two panels. Data is shown as average of all CpG values in each amplicon (as detailed in Supplementary Figure 6) and error bars represent the standard deviation.

Article Snippet: To perform the screen we obtained the TRC lentiviral mouse kinase shRNA library from the Broad Institute.

Techniques: Knockdown, Expressing, Generated, Western Blot, Positive Control, Transduction, Control, Plasmid Preparation, Cell Culture, shRNA, Marker, Immunofluorescence, DNA Methylation Assay, Amplification, Standard Deviation

Journal: Epigenetics

Article Title: An shRNA kinase screen identifies regulators of UHRF1 stability and activity in mouse embryonic stem cells

doi: 10.1080/15592294.2022.2044126

Figure Lengend Snippet: Knockdown of Csnk2b rescues expression of UHRF1 in 2i conditions. a) Csnk2b KD ESCs grown in 2i conditions display an altered morphology, with cells failing to form the domed, round colonies characteristic of 2i cells. Data is shown for Serum/LIF KD and 2i Csnk2b KD ESCs generated by shRNA1. b) Western blot analysis for UHRF1 upon knockdown of Csnk2b. Analysis was performed for cell lines generated from the two shRNAs producing a successful knockdown. The Western blot was performed in duplicate along with a positive control (Serum/LIF ESCs), as well as cells transduced with a non-silencing control vector grown in 2i conditions (NS 2i). Densitometry analysis was performed displaying the density of the UHRF1 band relative to the GAPDH band, error bars represent the minimum and maximum values from 2 technical replicates. The results show that knock-down of Csnk2b prevents degradation of UHRF1 in cells cultured in 2i conditions, and therefore replicates the results of the shRNA screen. c) Six candidate genes whose expression is known to change in Serum/LIF to 2i were measured in Csnk2b KD ESCs shRNA1, grown in both Serum/LIF and 2i conditions. Data are presented as 2− ΔCt with Gapdh serving as a loading control. Error bars represent the standard error of the mean from 3 technical replicates, * denotes P value < 0.05. d) Expression of the pluripotency marker NANOG as measured by immunofluorescence. Data is shown for Csnk2b KD ESCs grown in both Serum/LIF and 2i. Data is shown for 2 representative images, and DAPI was used as a nuclear stain. Data is for the knockdown cell line produced by shRNA1. e) DNA methylation in each KD cell line is shown in the two panels. Data is shown as average of all CpG values in each amplicon (as detailed in Supplementary Figure 6) and error bars represent the standard deviation in 3 technical replicates.

Article Snippet: To perform the screen we obtained the TRC lentiviral mouse kinase shRNA library from the Broad Institute.

Techniques: Knockdown, Expressing, Generated, Western Blot, Positive Control, Transduction, Control, Plasmid Preparation, Cell Culture, shRNA, Marker, Immunofluorescence, Staining, Produced, DNA Methylation Assay, Amplification, Standard Deviation