Journal: Nucleic Acids Research

Article Title: Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency

doi: 10.1093/nar/gkad456

Figure Lengend Snippet: Small RNA-seq analysis of different pegRNA species bound to the Prime editor in HEK293T cells. ( A ) Schematic for the small RNA-seq library preparation. Briefly, HEK293T cells were transfected with plasmids encoding one of two effectors (SpCas9 or PEmax), and one guide RNA (sgRNA, pegRNA or epegRNA). Cells were harvested after 2 days, crosslinked and then lysed for total RNA isolation. To sequence the bound pegRNA or epegRNA population, the SpCas9 or PEmax protein (containing 3xHA-tag) was immunoprecipitated then crosslinking was reversed to purify the bound RNA. This was followed by 3’ DNA adapter ligation (3’ adapter contains 15 bp UMI sequence) to the purified RNA, cDNA synthesis and two rounds of PCR to add sequencing adapters. The final library was deep sequenced and analyzed. A detailed protocol is present in the methods section. ( B ) Bulk or effector-bound RNA species present from each treatment group. ‘Bulk’ indicates sequencing of the sgRNA/pegRNA present in the cell without IP pulldown to examine the sgRNA/pegRNA 3’ sequence lengths irrespective of whether it is bound to SpCas9 or PEmax. The length of the PBS in the pegRNA (7 or 13 nt) is indicated in the name. Small RNAs were categorized into six species based on the length of 3’ truncation: full-length pegRNA, pegRNA with truncated but potentially functional PBS (≥7 nt remaining), pegRNA with truncated likely insufficient PBS (<7 nt), pegRNA with truncated RTT, and pegRNA with truncated sgRNA scaffold. Abundance of each RNA species was calculated based on UMIs incorporated into the 3’ adaptor from the small RNA-seq library (see for IGV plots). ( C ) RNP-mediated PE3 editing efficiencies in mCherry reporter cell line with different ratio of pegRNA:nicking sgRNA. The amount of PEmax protein (50 pmol) and pegRNA (200 pmol; IDT) was held constant while increasing the amount of nicking sgRNA (IDT) delivered by electroporation. Frequency of mCherry positive cells was quantified by flow cytometry 72 h following treatment. One-way ANOVA was used to compare all the groups for each graph, PE2 was used as a control column for multiple comparisons. ns stands for P > 0.05, * indicates P ≤ 0.05,** indicates P ≤ 0.01, and **** indicates P ≤ 0.0001 (also see Supplementary table). (D, E) RNP-mediated PE3 editing efficiencies at the specified positions for ( D ) FANCF (+5 G to T) and ( E ) HEK4 (+5 G to T) loci in HEK293T cells. The amount of PEmax protein (50 pmol) and pegRNA (200 pmol; IDT) was held constant while increasing the amount of nicking sgRNA (from IDT) delivered by electroporation. Editing efficiency reflects the frequency of sequencing reads from amplicon deep sequencing that contain the intended edit or others (indels and imprecise prime editing) among all sequencing reads. Values and error bars reflect mean ± s.d. of n = 3 independent biological replicates. One-way ANOVA was used to compare the intended edit across all the groups for each graph, PE2 was used as a control column for multiple comparisons. ns indicates P > 0.05, * indicates P ≤ 0.05,** indicates P ≤ 0.01, and **** indicates P ≤ 0.0001 (also see Supplementary table).

Article Snippet: Backbone plasmids used for pegRNA and sgRNA cloning are available from Addgene (#122089).

Techniques: RNA Sequencing, Transfection, Isolation, Sequencing, Immunoprecipitation, Adapter Ligation, Purification, cDNA Synthesis, Functional Assay, Electroporation, Flow Cytometry, Control, Amplification

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Calcium/calmodulin-dependent protein kinase kinase 2 mediates pleiotropic effects of epidermal growth factor in cancer cells.

doi: 10.1016/j.bbamcr.2022.119252

Figure Lengend Snippet: Fig. 1. CaMKK2 and PDK1 mediate Akt phosphorylation stimulated by EGF. (A) OVCAR-3 cells were transfected with non-specific (NS), PDK1 or CaMKK2 siRNAs for 2 days as shown. Cells were then serum-starved for 30 min, followed by EGF treatment (100 nM) for 5 min. Samples were analyzed by Western Blotting. Quanti fication of Akt phosphorylation at T308 and S473 is shown in (B) and (C) respectively (n = 4). Results are normalized to vehicle control and shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: AZD2014 (#A11303) was purchased from AdooQ. myc-Rictor was from Dr. David Sabatini, obtained from Addgene (plasmid #11367). p-Akt (T308) (#13038), p-Akt (S473) (#4060), panAkt (#4691), PDK1 (#13037), p-AMPK (T172) (#2525) and AMPK antibodies (#2603) were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Transfection, Western Blot, Control

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Calcium/calmodulin-dependent protein kinase kinase 2 mediates pleiotropic effects of epidermal growth factor in cancer cells.

doi: 10.1016/j.bbamcr.2022.119252

Figure Lengend Snippet: Fig. 2. Calcium and calmodulin regulation of Akt phosphorylation in response to EGF or the calcium ionophore, ionomycin. (A) OVCAR-3 cells were transfected with NS, PDK1 or CaMKK2 siRNAs for 2 days as shown. Cells were then serum starved for 30 min, followed by ionomycin treatment (1 μM) for 15 min. Samples were analyzed by Western Blotting. Quantification of Akt phosphorylation at T308 and S473 is shown in (B) and (C), respectively (n = 6). (D) OVCAR-3 cells were pre- treated with BAPTA-AM (10 μM) as shown during the 30 min serum starvation. Cells were then treated with EGF (100 nM) for 5 min or ionomycin (1 μM) for 15 min. Samples were analyzed by Western Blotting. Quantification of Akt phosphorylation at T308 and S473 is shown in (E) and (F), respectively (n = 3). (G) OVCAR-3 cells were pre-treated with W7 (10 μM) as shown during the 30 min serum starvation. Cells were then treated with EGF (100 nM) for 5 min or ionomycin (1 μM) for 15 min. Samples were analyzed by Western Blotting. Quantification of Akt phosphorylation at T308 and S473 is shown in (H) and (I), respectively (n = 6). (B, C, E, F, H, I) Results are normalized to control and shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: AZD2014 (#A11303) was purchased from AdooQ. myc-Rictor was from Dr. David Sabatini, obtained from Addgene (plasmid #11367). p-Akt (T308) (#13038), p-Akt (S473) (#4060), panAkt (#4691), PDK1 (#13037), p-AMPK (T172) (#2525) and AMPK antibodies (#2603) were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Transfection, Western Blot, Control

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Calcium/calmodulin-dependent protein kinase kinase 2 mediates pleiotropic effects of epidermal growth factor in cancer cells.

doi: 10.1016/j.bbamcr.2022.119252

Figure Lengend Snippet: Fig. 3. CaMKK2 neither binds to, nor is regulated by, PIP3 but is activated by Ca2+/CaM. (A) Schematic diagram of the phosphoinositide (PIP) array (Echelon Biosciences) used in these experiments. (B) The PIP array was incubated with baculovirus-expressed-PDK1 or CaMKK2 in the presence or absence of Ca2+/CaM. Proteins binding on the array were detected by blotting with PDK1 and CaMKK2 antibodies. (C) CaMKK2 kinase activity was measured using a peptide substrate derived from the Akt sequence flanking T308 and [γ-32P] ATP in the presence or absence of PIP3 and/or Ca2+/CaM (n = 3) (D) Baculovirus-expressed CaMKK2 and Akt were incubated under phosphorylating conditions in the presence or absence of PIP3 and/or Ca2+/CaM. Akt phosphorylation at T308 was analyzed by Western Blotting. (E) OVCAR-3 cells were transfected with PDK1 or CaMKK2 siRNAs for 2 days. Cells were then pre-treated with BAPTA-AM (10 μM) as shown during the serum starvation for 30 min, followed by EGF treatment (100 nM) for 5 min. Samples were analyzed by Western Blotting.

Article Snippet: AZD2014 (#A11303) was purchased from AdooQ. myc-Rictor was from Dr. David Sabatini, obtained from Addgene (plasmid #11367). p-Akt (T308) (#13038), p-Akt (S473) (#4060), panAkt (#4691), PDK1 (#13037), p-AMPK (T172) (#2525) and AMPK antibodies (#2603) were purchased from Cell Signaling Technology.

Techniques: Incubation, Binding Assay, Activity Assay, Derivative Assay, Sequencing, Phospho-proteomics, Western Blot, Transfection