Journal: bioRxiv

Article Title: Spatio-temporal analysis of the innate immune response to cytoplasmic dsDNA using a novel cGAMP biosensor

doi: 10.1101/2024.06.10.598238

Figure Lengend Snippet: A , B , Live cell imaging analyses of HEK293T and HFF-1 cGAMP-biosensor cells treated with 2 μM diABZI. C , IFNb-Luc reporter assays in HEK293T cells transfected with empty plasmid or wt STING, or HEK293T cGAMP-biosensor cells, and treated with the indicated concentrations of the STING agonist diABZI. Note that HEK293T do not express endogenous cGAS or STING.

Article Snippet: To generate the HeLa and HEK293T cGAMP biosensor cells, the cGAMP biosensor was cloned into the PiggyBAC backbone plasmid (Biocat PB510B-1-SBI).

Techniques: Live Cell Imaging, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening

doi: 10.1038/s41467-023-43790-2

Figure Lengend Snippet: a Survival curves of irradiated B6 mice inoculated with B-ALL cells and treated with indicated CAR-T cell type. Both significant P values displayed = 0.0035. Bioluminescence imaging four days after adoptive cell transfer (ACT) in b untreated mice, c mice treated with 10 7 control CAR-T cells, d treated with 7 × 10 6 anti-mCD19 CAR-T cells, or e treated with 10 7 anti-mCD19 CAR-T cells, and quantified in ( f ), both significant P values displayed <0.001; n = 10 mice per group from two independent experiments). g In vitro cytotoxicity assays show significant depletion of tumor cells along with h target epitope loss when B-ALL cells are treated with anti-mCD19 CAR-T cells at increasing effector to target cell (E:T) ratios. i Concomitantly, CAR-T cells expand and j release IFN γ when co-cultured with mCD19+ B-ALL cells. For (g–j), n = 3 biologically independent samples per group except for (j) where n = 6 biologically independent samples for the B-ALL only group. k – m Experiments to determine the appropriate CAR-T cell dose for either the bone marrow (BM) or spleen (SP) using flow cytometry analysis. Peripheral blood (PB) was also assessed (n = 4 mice per group). l Target epitope loss and m significant CAR-T cell persistence was also observed in all of the organs harvested from mice treated with anti-mCD19 CAR-T cells. n Schematic showing the overall design and o lentiviral backbone used to create the SKY library. p Retroviral vectors encoding the 1D3 single chain variable fragment (scFv) targeting mCD19 (top) and the 3C10 scFv targeting hEGFRvIII (bottom). q Diagram of screening layout. Data are mean ± s.e.m. All experiments were repeated at least twice with representative data shown. The significance for survival experiments was determined using log-rank tests. For all other experiments, significance is determined using unpaired two-sided student’s t-tests with Bonferroni correction for multiple comparisons, or using one-way ANOVA with Tukey’s correction for multiple comparisons when more than two groups were compared. * P < 0.05; ** P <  0.01; *** P <  0.001; **** P <  0.0001. Exact P values for each comparison shown in ( g – j ) and ( k – m ) can be found in Supplementary Data .

Article Snippet: HEK293T (293 T) cells were cultured in DMEM with L-glutamine and sodium pyruvate (Corning, 10-013-CM) supplemented with 10% FBS.

Techniques: Irradiation, Imaging, Control, In Vitro, Cell Culture, Flow Cytometry, Retroviral, Comparison

Journal: Nature Communications

Article Title: Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening

doi: 10.1038/s41467-023-43790-2

Figure Lengend Snippet: a Schematic of the data analysis and screen hit discovery workflow. b , c Waterfall plots of log-fold changes of the representation of sgRNAs against different genes in anti-mCD19 CAR-T cell treated animals b or cells c compared to anti-hEGFRvIII CAR-T cell treated animals b or cells c at indicated doses (15 m or 1.5 × 10 7 CAR-T cells, 10 m or 1 × 10 7 CAR-T cells, effector to target (E:T) ratios of 1:2, or 1:10). For in vivo screens, the organ from which guide-bearing B-ALL cells were collected is also indicated. Genes are ranked by the average log 2 fold changes (L2FC) of all sgRNAs against each gene, and point sizes are proportional to the magnitude (absolute value) of L2FC. Waterfall plots display relative biological effect of each hit versus guide ranks. d Relative enrichment/depletion of individual guides against top depleting genes Ifngr1 , Jak2 , Stat1 , and Qa-1 b are shown in each arm of the screen. Guide RNAs against Cd19 are also shown as an indicator of CAR-T treatment pressure. Enriched gRNAs are shown in red. Depleted gRNAs are shown in blue. e Venn diagrams showing overlap of top hits from the primary in vitro and in vivo screens. f A strip chart representation of the L2FC of sgRNAs targeting genes in the Biocarta_IFNG_Pathway (shown in blue) upon treatment with Cd19 CAR-T cells in vitro (left two columns) or in vivo (right three columns). g (Left) A gprofiler plot showing the pathways targeted by the most enriched sgRNAs in the in vitro E:T of 1:10 treatment cohort. (Right) A table showing a list of pathways targeted by the most enriched sgRNAs. h Waterfall plots from the validation screen showing the representation of sgRNAs targeting different genes in anti-mCD19 CAR-T cell treated animals or cultured cells compared to control treated organs or cultured cells. Waterfall plots on top show sgRNAs targeting Cd19 and select interferon gamma pathway genes (in yellow). Waterfall plots below show the top ten depleted, or top five depleted/enriched sgRNAs in each context.

Article Snippet: HEK293T (293 T) cells were cultured in DMEM with L-glutamine and sodium pyruvate (Corning, 10-013-CM) supplemented with 10% FBS.

Techniques: In Vivo, In Vitro, Stripping Membranes, Biomarker Discovery, Cell Culture, Control

Journal: Nature Communications

Article Title: Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening

doi: 10.1038/s41467-023-43790-2

Figure Lengend Snippet: a Schematic showing the IFN γ R signaling pathway. b , c In vivo competitive assays demonstrate specific depletion of Cas9 + RH62 B-ALL cells lacking components of the IFNγR/JAK/STAT pathway and enrichment of B-ALL cells lacking mCD19 after treatment with anti-mCD19 CAR-T cells in b the bone marrow and c spleen. For b , c , n = 9 for the groups Ifngr1 sgRNA#1, Jak2 sgRNA#1 and 2, treated with anti-hEGFRvIII CAR-T cells; n = 8 for the groups Cd19 sgRNA#1 treated with anti-hEGFRvIII or anti-mCD19 CAR-T cells; n = 6 for the group Jak2 sgRNA#1 treated with anti-mCD19; n = 4 for the groups Ifngr1 sgRNA#1 and 2, Stat1 sgRNA#2, and lacZ sgRNA#1, treated with anti-mCD19 CAR-T cells; n = 5 for all other groups. d Kaplan-Meier curves showing survival in immunocompetent mice transplanted with B-ALL cells deficient in the indicated IFN γ R/JAK/STAT pathway member. e In vivo competitive assays demonstrate specific depletion of Cas9 + RH62 B-ALL cells lacking Ptpn2 or Fitm2 after treatment with anti-mCD19 CAR-T cells in the spleen. Data shown is from flow cytometry analyses examining the proportion of live B-ALL cells that are APC + and therefore, also guide-bearing. In these experiments, n = 5 mice in groups treated with anti-hEGFRvIII CAR-T cells and n = 3 mice in groups treated with anti-mCD19 CAR-T cells. f A Kaplan–Meier curve showing overall survival in leukemia-bearing mice treated with 2.5 × 10 6 control or anti-mCD19 CAR-T cells in the presence or absence of blocking IFN γ antibody. g A Kaplan-Meier curve of overall survival in leukemia-bearing mice treated with control or 2.5 × 10 6 anti-mCD19 CAR-T cells in the presence or absence of Ruxolitinib, a JAK1/2 inhibitor. In vivo competition assays were repeated three times. Survival experiments were completed twice. Pharmacologic studies using anti-IFN γ blocking antibodies or JAK inhibitor were completed twice. The significance of survival experiments was determined using log-rank tests. For all other experiments, significance was determined using unpaired two-sided student’s t-tests with Bonferroni correction for multiple comparisons. Data are mean ± s.e.m. * P < 0.05; ** P <  0.01; *** P <  0.001; **** P <  0.0001. Exact P values for each comparison shown in (b-d) and (f-g) can be found in Supplementary Data .

Article Snippet: HEK293T (293 T) cells were cultured in DMEM with L-glutamine and sodium pyruvate (Corning, 10-013-CM) supplemented with 10% FBS.

Techniques: In Vivo, Flow Cytometry, Control, Blocking Assay, Comparison

Journal: Nature Communications

Article Title: Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening

doi: 10.1038/s41467-023-43790-2

Figure Lengend Snippet: a Schematic showing known effects of the HLA-E/NKG2A/CD94 axis in human NK and CD8 + T-cells. b In vitro competitive assays where CD19-expressing H2-T23 −/− and CD19-expressing H2-T2 +/+ pancreas cancer cells were mixed in a 50:50 ratio and exposed to the indicated concentration of anti-mCD19 CAR-T cells. The log 2 fold change (L2FC) of H2-T23 −/− to control cells is shown. Cells were either pre-treated (right) with exogenous recombinant IFN γ or left untreated throughout the experiment (left). For the no T cells groups, n = 2 biologically independent samples; n = 5 for all other groups. c Immunocompetent (C57BL/6 J) mice transplanted with H2-T23 −/− cells show increased survival compared to immunocompromised (NSG) mice transplanted with the same H2-T23 −/− cells and treated with anti-mCD19 CAR-T cells (n = 5 mice per group). d Mean fluorescence intensity (MFI) of Qa-1 b -Brilliant Violet 786 (BV786) after in vitro CAR-T treatment of single cell clones (scc) deficient in IFN γ R/JAK/STAT pathway members at two different E:T ratios. Wildtype scc were also assayed, as shown. e Treatment schedule for the anti-NKG2A blocking antibody experiment. f A Kaplan-Meier curve showing overall survival in mice treated with a murine version of the anti-NKG2A antibody, monalizumab or a control antibody, concurrently with mCD19 or control hEGFRvIII CAR-T cells, as indicated. g Kaplan-Meier curves showing overall survival in mice treated concurrently with either anti-NK1.1 or isotype control antibodies along with either anti-mCD19, anti-hEGFRvIII, or no CAR-T cells. In this experiment, mice were pre-treated with anti-NK1.1 antibody one day prior to CAR-T therapy. The survival experiments and in vitro CAR-T treatment of H2-T23 −/− cells were completed at least twice and were paired each time. Pharmacologic experiments were completed once. The significance of survival experiments was determined using log-rank tests. For all other experiments, significance was determined using unpaired two-sided student’s t-tests with Bonferroni correction for multiple comparisons. Data are mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Exact P values for each comparison shown in ( b – d ) and ( f – g ) can be found in Supplementary Data .

Article Snippet: HEK293T (293 T) cells were cultured in DMEM with L-glutamine and sodium pyruvate (Corning, 10-013-CM) supplemented with 10% FBS.

Techniques: In Vitro, Expressing, Concentration Assay, Control, Recombinant, Fluorescence, Clone Assay, Blocking Assay, Comparison

Journal: Nature Communications

Article Title: Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening

doi: 10.1038/s41467-023-43790-2

Figure Lengend Snippet: a PCA plot of bulk RNA-seq profiles of bone marrow (BM) and spleen (SP) samples collected from mice treated with either anti-mCD19 or anti-hEGFRvIII (control) CAR-T cells. b Heatmap of genes differentially expressed between anti-mCD19 and anti-hEGFRvIII CAR-T cell-treated samples. Scores of genes from the screen are shown next to each gene’s expression profile (column). Top depleting sgRNAs in the BM and SP are labeled. c Top Hallmark gene sets enriched among genes overexpressed in mCD19 CAR-T cell therapy compared to control treated BM or SP samples. Bar graphs show normalized enrichment scores for each of the top 5 enriched pathways. Enrichment analysis was performed using GSEA and all pathways shown have FDR < 0.05. d 2-dimensional UMAP plots of single cell gene expression profiles collected from mice treated with either anti-mCD19 or anti-hEGFRvIII CAR-T cells. Data are collected from n = 3 mice per treatment group. Left: cells color-coded by clusters discovered through unsupervised clustering. Right: cells color-coded by treatment and tissue groups. e Participation of cells from each tissue/treatment group in each cluster. Bar graphs show percentage of cells in each cluster belonging to each tissue/treatment group (anti-mCD19 CAR-T cell treated BM or SP labeled CD19BM and CD19SP, respectively; anti-hEGFRvIII control CAR-T treated BM or SP labeled V3BM and V3SP, respectively). Clusters where there is substantial enrichment of cells under anti-mCD19 CAR-T therapy are marked with a black box underneath the bar plots. f Hallmark gene sets enriched in the 4 treatment-specific clusters as labeled in ( e ). g Dot plot showing relative expression patterns of cluster 2 genes which are also among top depleting sgRNAs (L2FC < −1.75) in the BM samples of the in vivo screen. Dot size is proportional to the percentage of cells in each cluster expressing each gene and dot color indicates average expression of each gene in each cluster. h UMAP plots showing expression of Qa-1 b and Stat1 in single cell samples, with cells color-coded by expression levels.

Article Snippet: HEK293T (293 T) cells were cultured in DMEM with L-glutamine and sodium pyruvate (Corning, 10-013-CM) supplemented with 10% FBS.

Techniques: RNA Sequencing, Control, Expressing, Labeling, Gene Expression, In Vivo

Journal: Nature Communications

Article Title: Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening

doi: 10.1038/s41467-023-43790-2

Figure Lengend Snippet: a Normalized expression of a sensitizer gene signature (composed of top depleting genes in the BM arms of our screen) in pre-treatment leukemia samples from patients who are complete responders (CR) or non-responders (NR) to anti-hCD19 CAR-T therapy. The five number summaries of the CR and NR boxplots are 8.45, 9.09, 9.36, 9.72, 10.20, and 9.05, 9.47, 9.87, 10.08, 10.15, respectively. b Normalized expression of a JAK/STAT/MHC-I resistance signature in the same pre-treatment leukemia samples shown in ( a ). The five number summaries of the CR and NR boxplots are 8.77, 10.01, 10.65, 10.98, 11.62, and 9.87, 10.64, 11.24, 11.50, 11.78, respectively. For (a-b), the plots are graphed from minima to maxima and all data points are overlaid. c Normalized expression of the immune checkpoint blockade resistance-associated interferon-stimulated genes (ISG.RS) signature in B-ALL cells (after CAR-T failure) residing in expression cluster 2 or other cells. The ISG.RS signature is associated with poor outcomes in patients with large B-cell lymphoma treated with anti-CD19 CAR-T cells. A two-sided Wilcoxon rank-sum test was performed to compare the two distributions shown. An exact P value cannot be numerically determined due to ties in rank sum tests. A numerically approximate P value is shown. The plots are graphed in the Tukey method. The five-number summary for cluster 2 cells (left) is 0.036, 0.22, 0.28, 0.36, 0.57, and 0, 0.12, 0.18, 0.24, 0.42 for all other cells (right). d A final model for how high IFN γ R/JAK/STAT signaling in tumor cells can promote resistance to CAR-T therapy via the upregulation of the NK and CD8 + T-cell inhibitory molecule Qa-1b, the murine homolog of HLA-E. Except where indicated, significance is determined using unpaired two-sided student’s t-tests with Bonferroni correction for multiple comparisons. Data are mean ± s.e.m. * P < 0.05; ** P <  0.01; *** P <  0.001; **** P <  0.0001.

Article Snippet: HEK293T (293 T) cells were cultured in DMEM with L-glutamine and sodium pyruvate (Corning, 10-013-CM) supplemented with 10% FBS.

Techniques: Expressing

Journal: bioRxiv

Article Title: Quantitative design of cell type-specific mRNA stability from microRNA expression data

doi: 10.1101/2024.10.28.620728

Figure Lengend Snippet: ( A ) We measured constructs containing two repeats of a microRNA target site. The sequence is identical to the same constructs in Library 1. ( B ) Gating strategy on SSC-A and FSC-A for HEK293T cells. ( C ) The gating for transfected cells using an mCherry-expressing transfection control. ( D ) GFP fluorescence distribution for one replicate in HEK293T after filtering for transfection using the mCherry signal. The negative control is shown without mCherry gating. ( E ) Median fluorescence normalized to the median fluorescence of the positive control for two different microRNA target types versus the transfected plasmid concentration. The result is largely independent of plasmid concentrations when using the median fluorescence. N=3 ( F ) Relationship between the stability measured for Library 1 for our high-throughput stability data and the median GFP fluorescence level normalized to the median fluorescence of the positive control measured using flow cytometry for two different cell lines (HEK293T, HeLa). N=3

Article Snippet: We measured 200 μl of cell suspension either undiluted (HeLa) or diluted 1:1 in PBS (HEK293T) using an Attune NxT Flow Cytometer (ThermoFisher).

Techniques: Construct, Sequencing, Transfection, Expressing, Control, Fluorescence, Negative Control, Positive Control, Plasmid Preparation, Concentration Assay, High Throughput Screening Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Quantitative design of cell type-specific mRNA stability from microRNA expression data

doi: 10.1101/2024.10.28.620728

Figure Lengend Snippet: ( A ) Ago1 and Ago2 expression according to protein atlas data versus the fitted scaling factor for individual target sites. Ago1 expression is negatively and Ago2 expression is positively correlated with the scaling factor. We used Ago2 expression as a proxy for the total amount of slicing-competent miRNA in a cell. ( B ) Left: The distribution of baseline stabilities for 51 different 3’UTRs (Methods) without miRNA target sites. Because normalization is performed relative to the stability of the main context, shown stabilities are also relative to the stability of the main context sequence. Right: The inverse of the geometric mean of the stabilities shown on the left versus the fitted scaling factor. This value approximates the relative stability of the main context across cell lines. ( C ) Dividing the relative Ago2 expression normalized by the HEK293T value by the geometric mean of the context stabilities yields a good estimate of the overall scaling factor.

Article Snippet: We measured 200 μl of cell suspension either undiluted (HeLa) or diluted 1:1 in PBS (HEK293T) using an Attune NxT Flow Cytometer (ThermoFisher).

Techniques: Expressing, Sequencing

Journal: FEBS letters

Article Title: Modulation of GSK3β autoinhibition by Thr-7 and Thr-8

doi: 10.1002/1873-3468.12990

Figure Lengend Snippet: Alanine mutations of highly conserved Thr-7 and Thr-8 on the N-terminus of GSK3β impairs Ser-9 phosphorylation in vivo and GSK3β autoinhibition by insulin. (A) In the N-terminus of human GSK3β, highly conserved Thr-7 and Thr-8 residues are located adjacent to the inhibitory phosphorylation site Ser-9 within the AKT substrate consensus sequence R-P-R-T7-T8-S9-F. (B) HA-GSK3β wild type (WT) and T-to-A mutant (T7A, T8A, S9A) proteins were expressed in HEK293T cells, purified by anti-HA agarose beads, and incubated in vitro with cold ATP and His-Akt1 at 30°C for various time points as indicated. Total and phospho-S9 specific signals of HA-GSK3β were detected on Western blot. Phospho-S9 signals were quantified and normalized against total GSK3β signals. (C) HA-GSK3β (WT, T7A, T8A) proteins were expressed in HEK293T cells under normal fed condition. Whole cell extracts were Western blotted for total and phospho-S9 signals of endogenous and HA-tagged GSK3β. Shown are data of duplicates. (D) HA-GSK3β (WT, T7A) proteins were expressed in HEK293T cells under normal fed condition. After anti-HA immunoprecipitation, total and phospho-S9 signals of HA-GSK3β as well as endogenous AKT co-immunoprecipitated with HA-GSK3β were analyzed by Western blot. Shown are data of triplicates. (E) HA-GSK3β WT or mutant (T7A, T8A) proteins were expressed in HEK293T cells. After overnight starvation in 0.1% low-serum medium, cells were treated with 100 nM insulin for various time ponits as indicated. Phospho-S9 specific and total GSK3β signals in whole cell extracts (WCE) or in anti-HA immunoprecipitates were analyzed by Western blot. Shown are representative data from three independent experiments. (F) HA-GSK3β WT and mutant (T7A, T8A, S9A) proteins were expressed in HEK293T cells. After starvation and insulin stimulation (100 nM, 60 min), cells were lysed and HA-GSK3β proteins were affinity-purified on anti-HA agarose beads. HA-GSK3β WT proteins purified from unstimulated cells served as the control. In vitro kinase assay was conducted to assess GSK3β activity toward p-GS peptide substrates. Shown are GSK3β activities relative to the control, mean ± SD, n=3 independent experiments, *P<0.01.

Article Snippet: HEK293T cells were transfected using either a calcium phosphate protocol as described in [ 30 , 32 ] or the lipofectamine 2000 reagent following the manufacturer’s instruction (Invitrogen, Carlsbad, CA, USA).

Techniques: Phospho-proteomics, In Vivo, Sequencing, Mutagenesis, Purification, Incubation, In Vitro, Western Blot, Immunoprecipitation, Affinity Purification, Control, Kinase Assay, Activity Assay

Journal: FEBS letters

Article Title: Modulation of GSK3β autoinhibition by Thr-7 and Thr-8

doi: 10.1002/1873-3468.12990

Figure Lengend Snippet: The inhibitory effect of the T7A or T8A mutation on GSK3β Ser-9 phosphorylation cannot be abolished by inhibition of phosphatase activities. (A) HEK293T cells were starved in low-serum medium overnight, and then treated with 100 nM insulin for 30 min followed by 50 nM Calyculin A for 30 min prior to lysis. Whole cell extracts were blotted for both total and phosphorylation signals of mTOR, S6K, GSK3, and ERK. (B) HA-GSK3β WT or mutant (T7A, T8A) proteins were expressed in HEK293T cells. After overnight starvation in 0.1% low-serum medium, cells were treated with 100 nM insulin for 30 min followed by 50 nM Calyculin A for 30 min prior to lysis. HA-GSK3β proteins were isolated from whole cell lysate by anti-HA immunoprecipitation. Phospho-S9 and total GSK3β signals in anti-HA immunoprecipitates were analyzed by Western blot. Note that Calyculin A is a potent, cell permeable inhibitor of PPP family phosphatases including PP1, PP2A, PP4, PP5, and PP6.

Article Snippet: HEK293T cells were transfected using either a calcium phosphate protocol as described in [ 30 , 32 ] or the lipofectamine 2000 reagent following the manufacturer’s instruction (Invitrogen, Carlsbad, CA, USA).

Techniques: Mutagenesis, Phospho-proteomics, Inhibition, Lysis, Isolation, Immunoprecipitation, Western Blot

Journal: FEBS letters

Article Title: Modulation of GSK3β autoinhibition by Thr-7 and Thr-8

doi: 10.1002/1873-3468.12990

Figure Lengend Snippet: ICK phosphorylates GSK3β Thr-7 in vitro and in vivo. (A) HA-GSK3βKD (kinase dead mutant) was expressed in HEK293T cells and purified by anti-HA agarose beads. Purified HA-GSK3βKD substrates were incubated in vitro with 32γP-ATP and His-AKT or His-ICK1-291. Shown here is the 32P autoradiograph. (B) A non-radioactive kinase assay was conducted in vitro using wild type HA-GSK3β proteins as the substrate. Phospho-T7/S9 signals on HA-GSK3β were detected by phosphosite-specific antibodies on Western blot. (C) HA-GSK3β (WT, T7A, T8A, S9A) proteins were expressed in HEK293T cells, purified by anti-HA agarose beads, and incubated in vitro with cold ATP and His-ICK1-291. Total and phosphosite specific signals of HA-GSK3β were detected on Western blot. (D) Wild type (WT) or kinase dead (KD) GST-ICK proteins were co-expressed with WT or T7A mutant HA-GSK3β proteins in HEK293T cells. Total and phospho-T7 signals on purified HA-GSK3β proteins were detected on Western blot. (E) RIE-Ras12V cells were infected with lentivirus expressing either an validated ICK-specific shRNA (shICK) or the control shRNA (shCTL). Phospho-GSK3β T7 specific and total GSK3β and ICK signals were shown on Western blot.

Article Snippet: HEK293T cells were transfected using either a calcium phosphate protocol as described in [ 30 , 32 ] or the lipofectamine 2000 reagent following the manufacturer’s instruction (Invitrogen, Carlsbad, CA, USA).

Techniques: In Vitro, In Vivo, Mutagenesis, Purification, Incubation, Autoradiography, Kinase Assay, Phospho-proteomics, Western Blot, Infection, Expressing, shRNA, Control