sik2 protein  (Proteintech)

Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: Age-dependent downregulation of SIK2 was observed in the middle-aged AD transgenic mouse models and AD patients. a Bioinformatics analysis revealed reduced SIK2 expression in the temporal cortex of AD patients compared to controls. b Sik2 mRNA levels in the hippocampus of 2-, 5-, 8-, and 10-month-old WT and 5 × FAD mice ( n = 6/group), assessed by qPCR. c Quantification of SIK2 protein levels in the hippocampus of 2-, 5-, 8-, and 10-month-old WT and 5 × FAD mice ( n = 5/group), assessed by western blot. d Representative immunoblots of SIK2 in the dorsal hippocampus of 2-, 5-, 8-, and 10-month-old WT and 5 × FAD mice. e, f Representative immunoblots and quantification of SIK2 in the hippocampus of 2-, 5-, 8-, and 10-month-old WT and 5 × FAD mice ( n = 3/group). g, h MWM performance of WT and 5 × FAD mice. Escape latency during training trials (1–7 days) ( g ). Percentage of time spent in the target quadrant during the probe trial (day 8) ( h ). i Number of platform crossings during the probe trial (day 8). j, k Linear regression analysis was performed to assess the relationship between hippocampal SIK2 protein levels and cognitive performance (percentage of time in the target quadrant ( j ) and number of platform crossings ( k )) in WT ( n = 5) and 5 × FAD mice ( n = 5). The coefficient of determination ( R 2 ) and the P value from the F-test of the overall fit are shown on the graphs. The solid line represents the line of best fit. l SIK2-specific RNA probes combined with NeuN immunofluorescence in the dentate gyrus (DG) of WT and 5 × FAD mice. Scale bar, 50 μm. m Quantification of SIK2 intensity in DG NeuN⁺ cells ( n = 3/group). n Double-label immunofluorescence showing SIK2 (red) and NeuN (green) colocalization in the DG region of WT and 5 × FAD mice. Scale bar, 50 μm. o Quantification of SIK2 intensity in DG NeuN⁺ cells ( n = 3/group). Data are expressed as mean ± SEM. Statistical significance was calculated by unpaired Mann–Whitney test ( i ), unpaired two-tailed t -test ( a, h, m, o ), linear regression analysis ( j, k ), one-way ANOVA ( b, c, f ), and two-way ANOVA ( g ) followed by the Tukey’s post-hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Transgenic Assay, Expressing, Western Blot, Immunofluorescence, MANN-WHITNEY, Two Tailed Test

Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: SIK2 alleviates the cognitive impairment and enhances the synaptic plasticity in middle-aged 5 × FAD mice. a–e MWM performance of WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice. Escape latency during training trials (1–7 days) ( a ) and probe trial (day 8) ( b ). Platform crossings ( c ), percentage of time in the target quadrant ( d ), and swimming speed ( e ) during the probe trial (day 8). Sample sizes: n = 21 (WT-control), n = 22 (WT-SIK2), n = 19 (5 × FAD-control), n = 19 (5 × FAD-SIK2). f, g Long-term potentiation (LTP) recordings in hippocampal CA1 regions. High-frequency stimulation (HFS) was applied, and fEPSP amplitudes were quantified during the last 10 min ( g ). Sample sizes: n = 3 mice, 7 slices per group. h Ultrastructural analysis of synapses in hippocampal CA1 region via TEM. sv, synaptic vesicle; sc, synaptic cleft; PSD, postsynaptic density. Scale bars: 1 µm (top images), 250 nm (bottom images). i, j Quantitative analysis of PSD thickness and SC width ( n = 2 mice, 5 images per mouse). k-m Dendritic morphology of CA1 pyramidal neurons. Representative images ( k ) and Sholl analysis of branch intersections ( l-m ). Scale bars, 500 µm (upper), 50 µm (middle), 10 µm (lower). Sample size: n = 7 dendrites from 3 mice per group. n, o MAP2 immunofluorescence staining in the CA1 region. Representative images ( n ) and quantitative analysis ( o ). Scale bar, 50 µm. p, q Western blot analysis of SIK2, SYN, PSD95, and BDNF in dorsal hippocampal lysates. Representative immunoblots ( p ) and quantitative analyses ( q ) ( n = 6 per group). Data are expressed as mean ± SEM. Statistical significance was calculated by two-way ANOVA ( b, c-g , i, j, l, m, o, q ), and three-way ANOVA ( a ) followed by the Tukey’s post-hoc test, and Scheirer-Ray-Hare test followed by the Dunn’s post-hot test ( c ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, # P < 0.05, ## P < 0.01, & P < 0.05

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Control, Immunofluorescence, Staining, Western Blot

Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: SIK2 reduces Aβ deposition in 5 × FAD mice. a, b Representative immunoblots and quantitative analyses of SIK2, Aβ (6E10), APP, and BACE1 in the dorsal hippocampus of WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice ( n = 6/group). c, d Representative immunoblots and quantitative analyses of SIK2, Aβ (6E10), APP, and BACE1 in the dorsal hippocampus of WT-control, WT-shSIK2, 5 × FAD-control, and 5 × FAD-shSIK2 mice ( n = 6/group). e, g Immunofluorescence staining for Aβ (6E10) in the dentate gyrus (DG) of WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice. Representative images ( e ) and quantification ( g ) ( n = 3/group). Scale bar, 50 µm. f, h Immunofluorescence staining for Aβ (6E10) in the DG of WT-control, WT-shSIK2, 5 × FAD-control, and 5 × FAD-shSIK2 mice. Representative images ( f ) and quantification ( h ) ( n = 3/group). Scale bar, 50 µm. i, j ELISA quantification of soluble and insoluble Aβ 1–42 levels in hippocampal homogenates of WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice ( n = 4/group). k, l ELISA quantification of soluble and insoluble Aβ 1–42 levels in hippocampal homogenates of WT-control, WT-shSIK2, 5 × FAD-control, and 5 × FAD-shSIK2 mice ( n = 4/group). Data are expressed as mean ± SEM. Statistical significance was calculated by two-way ANOVA ( b-d , g-l ) followed by the Tukey’s post-hoc test. * P < 0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Western Blot, Control, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: SIK2 enhances autophagic flux in AD models. a KEGG pathway enrichment analysis showing significant activation of autophagy-related pathways in N2a-APP-SIK2 versus N2a-APP cells ( P = 0.0024, FDR < 0.05). b, c Representative immunoblots and quantitative analyses of SIK2, p62, LC3B, and APP in N2a, N2a-APP, N2a-SIK2, and N2a-APP-SIK2 cells ( n = 6). d, e Detection of lysosomal acidification in N2a, N2a-SIK2, N2a-APP, N2a-APP-SIK2, and N2a-APP-RAPA cells by mRFP-GFP-LC3 tandem fluorescence. Autophagosomes show double mRFP + GFP + signals (yellow spots), and functional autophagosomes showed mRFP + GFP - signals (red spots). Representative images ( d ) and quantization ( e ) ( n = 8–9/group). Scale bar, 10 μm. f, g Representative immunoblots and quantitative analyses of SIK2, p62, and LC3B in N2a-APP cells treated with chloroquine (CQ, 50 μmol/L, 12 h) or bafilomycin A1 (BafA1, 100 nmol/L, 12 h) ( n = 3 per group). h, i TEM images of autophagosomes (orange arrows) and autolysosomes (yellow arrows) in N2a, N2a-APP, N2a-SIK2, and N2a-APP-SIK2 cells. Scale bars, 1 µm. Quantitative analysis of autophagic vacuoles ( i ). j, k Representative immunoblots and quantitative analyses of SIK2, p62, and LC3B in WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice ( n = 6/group). l, m TEM images of autophagosomes (orange arrows) and autolysosomes (yellow arrows) in WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice. Scale bars, 500 nm. Quantitative analysis of autophagic vacuoles ( m ). Data are expressed as mean ± SEM. Statistical significance was calculated by unpaired two-way ANOVA ( c, e, g, i, k, m ) followed by the Tukey’s post-hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Activation Assay, Western Blot, Fluorescence, Functional Assay, Control

Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: SIK2 phosphorylates GABARAPL2 at Serine 72 to enhance autophagic flux. a-f Co-IP analysis of interactions between SIK2 and LC3A ( a ), LC3B ( b ), LC3C ( c ), GABARAP ( d ), GABARAPL1 ( e ), or GABARAPL2 ( f ) in N2a cells overexpressing SIK2-flag, using anti-flag antibodies. Asterisks indicate co-precipitated ATG8 bands. Hc: IgG heavy chain. g, h Co-IP analysis of interactions between LC3A, LC3B, LC3C, GABARAP, GABARAPL1, or GABARAPL2 and SIK2 in N2a cells overexpressing respective flag-tagged ATG8 family members, using anti-flag antibodies. i, j Representative immunoblots and quantification of SIK2, p62, and LC3B in N2a-APP cells with GABARAPL2 knockdown and SIK2 overexpression ( n = 3/group). k Schematic of SIK2 domain organization highlighting the kinase domain and LIR motif. Deletion mutants are illustrated below. l, m Co-IP analysis of interactions between GABARAPL2 and SIK2 deletion mutants (SIK2(1–290)-His, SIK2(1–550)-His, SIK2(1–650)-His, SIK2(1–931)-His) using anti-His antibodies. n, o Representative immunoblots and quantification of SIK2, p62, and LC3B in N2a-APP cells with SIK2 deletion mutants ( n = 3/group). p Competitive binding. Purified full-length SIK2 protein were incubated with GST-GABARAPL2 and increasing concentrations of LIR peptide and subjected to GST pull-down. q Quantification of competitive binding in ( p ). r Volcano plot of differentially phosphorylated sites in N2a-APP-SIK2 versus N2a-APP cells. s, t Representative immunoblots and quantification of GABARAPL2, p62, and LC3B in N2a-APP cells with GABARAPL2 mutants ( n = 3/group). u Representative immunoblots of GABARAPL2 and p-GABARAPL2(Ser72) in WT-control, WT-SIK2, 5 × FAD-control, and 5 × FAD-SIK2 mice ( n = 6/group). v The quantification of p-GABARAPL2 in the dorsal hippocampus ( n = 6/group). w, x N2a-APP cells overexpressing GABARAPL2-flag were transfected with control or SIK2 for 24 h. Immunoprecipitated with anti-flag antibody and probed with anti-p-Ser72-GABARAPL2 antibody by Western blot. Data are expressed as mean ± SEM. Statistical significance was calculated by unpaired two-tailed t -test ( x ), one-way ANOVA ( m, q ) and two-way ANOVA ( j, o, t, v ) followed by the Tukey’s post-hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Knockdown, Over Expression, Binding Assay, Purification, Incubation, Control, Transfection, Immunoprecipitation, Two Tailed Test

Journal: Translational Neurodegeneration

Article Title: SIK2-mediated phosphorylation of GABARAPL2 facilitates autophagosome–lysosome fusion and rescues neurodegeneration in an Alzheimer’s disease model

doi: 10.1186/s40035-025-00514-4

Figure Lengend Snippet: Phospho-GABARAPL2 at Ser72 alleviates the cognitive impairment of middle-aged 5 × FAD mice. a-e MWM performance of WT-control, 5 × FAD-control, 5 × FAD-PL2(WT), 5 × FAD-PL2(72A), and 5 × FAD-PL2(72E) mice. Escape latency during training trials (1–6 days) ( a ) and probe trial (day 7) ( b ). Platform crossings ( c ), percentage of time in the target quadrant ( d ), and swimming speed ( e ) during the probe trial (day 7). Sample sizes: n = 12 (WT-control), n = 10 (5 × FAD-control), n = 10 (5 × FAD-PL2(WT)), n = 10 (5 × FAD-PL2(72A)), n = 10 (5 × FAD-PL2(72E)). f, g Long-term potentiation (LTP) recordings in hippocampal CA1 regions. High-frequency stimulation (HFS) was applied, and fEPSP amplitudes were quantified during the last 10 min ( g ). Sample sizes: n = 3 mice, 7 slices per group. h, i Immunofluorescence staining for Aβ in the CA1 region. Representative images ( h ) and quantitative analysis ( i ). Scale bar, 50 µm. j, k Western blot analysis of GABARAPL2, p-GABARAPL2(Ser72), p62, LC3B, SYN, PSD95, and Aβ in dorsal hippocampal lysates. Representative immunoblots ( j ) and quantitative analyses ( k ) ( n = 6 per group). l, m TEM images of autophagic vacuoles. Representative images ( l ) and quantitative analysis ( m ). Orange arrows: double-membrane autophagosomes; yellow arrows: single-membrane autolysosomes with electron-dense contents. Scale bars, 500 nm. n KEGG pathway analysis of DEGs between 5 × FAD-control and 5 × FAD-PL2(72E) mice, showing the top 10 enriched pathways. o Proposed model illustrating how SIK2 regulates autophagosome–lysosome fusion and neurodegeneration in AD. Data are expressed as mean ± SEM. Statistical significance was calculated by two-way ANOVA ( b, d-g, i, k, m ), and three-way ANOVA ( a ) followed by the Tukey’s post-hoc test, and Scheirer-Ray-Hare test followed by the Dunn’s post-hot test ( c ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For in vitro binding assay, the SIK2 protein was incubated with GST (Proteintech, Ag0040, Wuhan, China) or GST-GABARAPL2 (Proteintech, Ag1155) protein which was immobilized on glutathione-conjugated Sepharose beads.

Techniques: Control, Immunofluorescence, Staining, Western Blot, Membrane

Journal: Cancer cell

Article Title: SIK2 is a centrosome kinase required for bipolar mitotic spindle formation that provides a potential target for therapy in ovarian cancer

doi: 10.1016/j.ccr.2010.06.018

Figure Lengend Snippet: A–D) SKOv3 cells were transfected with either non-targeting siRNA (A) or SIK2 siRNA (B) for 48 hr then monitored for 16 hr (one image every 5 minutes) using bright field phase-contrast microscopy. The time interval from nuclear envelope breakdown (0:00 in A and B) to anaphase onset (01:20 in control cells) was estimated. Arrows point to the cell under study. Time is shown as hours:minutes. Scale bar, 10 µm. In C Western blot confirmation of knockdown of SIK2 using 3 independent siRNAs compared to non-targeting siRNA control is shown. r-GST-SIK2; recombinant GST-tagged SIK2 was used on the same Western blot to confirm the antibody specificity. D) The mean ± s.e.m of the mitotic transition time (obtained as in A and B) is presented following control and SIK2 siRNA transfections. In E, SKOv3 cells (5000 cells per well) were reverse transfected in 96 well plates using either non-targeting controls or SIK2 siRNAs A, B and C for 24 hr and cells were then either a) fixed and stained using crystal violet to estimate the number of cells on the day of paclitaxel treatment (Day 0) or b) treated with either diluent or paclitaxel at 9 different concentrations. The latter group of cells was fixed and stained 72 hr following drug treatment. Growth of cells from day 0 to day 3 was estimated and the percentage paclitaxel-induced growth inhibition for each siRNA in relation to diluent treated cells transfected with the same siRNA was calculated and a least-squares fit was obtained to estimate the GI 50 as described in methods (Monks et al., 1991). Shown is the mean ± s.e.m from three replicates per concentration. In F the effect of depletion of SIK2 using siRNA C on paclitaxel response in three cell lines is shown. The experiment was conducted as in E. Shown is the mean ± s.e.m from six replicates per concentration. Also shown is the p value for the comparison between the GI 50 in cells transfected with non-targeting siRNA control v cells transfected with SIK2 siRNA. G) Microarray expression data for two probe sets representing SIK2 were used to generate Kaplan Meier survival curves for high expressing (defined as cancers with values above the median + (0.5 × median absolute deviation [MAD]) or low expressing (below the median − (0.5 × MAD) for 229 high-grade serous (HGS) ovarian cancers. See also Figure S2 and Movies Smov1–3.

Article Snippet: However, in G2, SIK2 expression resulted in a greater magnitude of C-Nap1 displacement (14.5% in SIK2 negative cells to 29.4% in SIK2 positive cells; p=0.004; Chi square test, n= 340, ).

Techniques: Transfection, Microscopy, Control, Western Blot, Knockdown, Recombinant, Staining, Inhibition, Concentration Assay, Comparison, Microarray, Expressing

Journal: Cancer cell

Article Title: SIK2 is a centrosome kinase required for bipolar mitotic spindle formation that provides a potential target for therapy in ovarian cancer

doi: 10.1016/j.ccr.2010.06.018

Figure Lengend Snippet: A) SKOv3 cells were fixed and stained for immunofluorescence (IF) using the indicated antibodies to show the co-localization between SIK2 and γ-tubulin. B) The co-localization of ectopically expressed myc-tagged SIK2 with γ-tubulin in SKOv3 cells is shown. Also note the peri-centrosomal expression of SIK2. Scale bar, 5 µm. See also Figure S3.

Article Snippet: However, in G2, SIK2 expression resulted in a greater magnitude of C-Nap1 displacement (14.5% in SIK2 negative cells to 29.4% in SIK2 positive cells; p=0.004; Chi square test, n= 340, ).

Techniques: Staining, Immunofluorescence, Expressing

Journal: Cancer cell

Article Title: SIK2 is a centrosome kinase required for bipolar mitotic spindle formation that provides a potential target for therapy in ovarian cancer

doi: 10.1016/j.ccr.2010.06.018

Figure Lengend Snippet: A) SKOv3 cells were transfected with myc/flag-tagged wild type SIK2 or myc/flag-tagged kinase mutant SIK2 (SIK2_mt) plasmid for 24 hr then cells were fixed and stained using anti-myc antibody (red), and anti-γ-tubulin antibody (green). Arrow heads show an example of CS (inter-centrosome distance > 2µm) following SIK2 transfection. B–C) Mean ± s.e.m of the percentage of cells with CS 24 hr following transfection using the indicated plasmids. Overexpression of NEK2, which is known to induce CS, was used as a positive control in this experiment. Experiments were performed at least in triplicates. Scale bar, 5 µm.

Article Snippet: However, in G2, SIK2 expression resulted in a greater magnitude of C-Nap1 displacement (14.5% in SIK2 negative cells to 29.4% in SIK2 positive cells; p=0.004; Chi square test, n= 340, ).

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Staining, Over Expression, Positive Control

Journal: Cancer cell

Article Title: SIK2 is a centrosome kinase required for bipolar mitotic spindle formation that provides a potential target for therapy in ovarian cancer

doi: 10.1016/j.ccr.2010.06.018

Figure Lengend Snippet: A–E)Cancer cells were transfected with siRNAs for 48 hr as indicated then fixed and stained. In A and B SKOv3 cells were stained using anti-γ-tubulin (red) and anti-phosphohistone H3 (green) to reveal the centosome position in mitosis in relation to chromosomes. Note the presence of examples from different mitosis phases in control cells (1–4; prophase, prometaphase, metaphase and anaphase, respectively) compared to the predominance of prometaphase cells following SIK2 KD. Also shown are examples of multipolar (I) and monopolar (II) centrosome positioning following SIK2 KD. Bar plots in B and D represent the mean ± s.d. of the percentages of cells at different stages of mitosis. Bar plots in C represent the mean prometaphase/metaphase ratio ± standard deviation. F–G) DMEL Drosophila cells were transfected using non-targeting control double stranded RNA (dsRNA) or dsRNAs targeting the Drosophila orthologue of SIK2. Examples of mitotic cells with failed centrosome separation are presented in F. In G, the mean ± s.d. of mitotic cells with failed centrosome separation from three independent experiments is shown. Scale bars, 10 µm. See also Figure S5.

Article Snippet: However, in G2, SIK2 expression resulted in a greater magnitude of C-Nap1 displacement (14.5% in SIK2 negative cells to 29.4% in SIK2 positive cells; p=0.004; Chi square test, n= 340, ).

Techniques: Transfection, Staining, Control, Standard Deviation

Journal: Cancer cell

Article Title: SIK2 is a centrosome kinase required for bipolar mitotic spindle formation that provides a potential target for therapy in ovarian cancer

doi: 10.1016/j.ccr.2010.06.018

Figure Lengend Snippet: A-SKOv3 cells were transfected with the indicated siRNAs for 72 hours then fixed and stained. The centrosome expression of PRKAR2A is present in control cells (arrow) but absent following KD of AKAP450 (I and II). B) Bar plots represent the mean ± s.d. of triplicate percentages of cells with CS following transfection with the indicated siRNAs. Depletion of AKAP450 resulted in a significant increase in CS while co-depletion of AKAP450 and SIK2 rescued this phenotype. Scale bar, 5 µm. See also Figure S6.

Article Snippet: However, in G2, SIK2 expression resulted in a greater magnitude of C-Nap1 displacement (14.5% in SIK2 negative cells to 29.4% in SIK2 positive cells; p=0.004; Chi square test, n= 340, ).

Techniques: Transfection, Staining, Expressing, Control

Journal: Cancer cell

Article Title: SIK2 is a centrosome kinase required for bipolar mitotic spindle formation that provides a potential target for therapy in ovarian cancer

doi: 10.1016/j.ccr.2010.06.018

Figure Lengend Snippet: A) SKOv3, ES2 and Hey cells were either transfected using non-targeting control siRNA or the indicated SIK2 siRNAs for 5 days. Cells were fixed and stained using crystal violet. Shown is the mean ± s.e.m. of 12 replicate values per siRNA transfection type from two independent experiments in SKOv3 cells. ** indicates p<0.001. Error bars for Hey and ES2 cells represent the s.e.m. of 6 replicate values per siRNA transfection type from two independent experiments. B) Typical cell cycle distributions are presented following transfection of SKOv3 cells with the indicated siRNAs. C) Cells were transfected using the indicated siRNA for 48 hr then treated with paclitaxel 100 nM for 24 hr, fixed and stained using anti-phosphohistone H3 antibody to calculate the mitotic index (MI) using flow cytometry. Shown is the mean MI (± s.d.) of two independent experiments. D) Cell lines were transfected with the indicated SIK2 siRNAs and control non-targeting siRNAs for 48 hr then harvested for analysis of protein expression using reverse phase protein arrays. Shown is the fraction of expression of AKT-pS437 and total AKT following SIK2 siRNA transfection relative to protein expression following non-targeting siRNAs. E) The expression of 139 proteins (Supplementary Table S4) was estimated using reverse phase protein arrays as in D following transfection of cancer cells using three SIK2 siRNAs and one non-targeting control in 3 cell lines; SKOv3, Hey and ES2. The expression of each of the 139 proteins was correlated with cancer cell growth that was measured in A. Each bar represents the coefficient of the correlation between the expression of each of the proteins in 9 cell lines (3 SIK2 siRNAs × 3 cell lines) and the growth of the cancer cell line from which the protein was measured. Positive values indicate that higher expression of a protein correlates with more cancer cell growth. Negative values indicate that higher expression correlate with poor growth. F) Shown is the regression lines for the correlation between the fraction of ovarian cancer cell proliferation following SIK2 depletion relative to non-targeting control and the fraction expression of AKT-pS437 or total AKT following SIK2 depletion relative to non-targeting controls. See also Figure S7 and Table S4.

Article Snippet: However, in G2, SIK2 expression resulted in a greater magnitude of C-Nap1 displacement (14.5% in SIK2 negative cells to 29.4% in SIK2 positive cells; p=0.004; Chi square test, n= 340, ).

Techniques: Transfection, Control, Staining, Flow Cytometry, Expressing

Journal: Cancer cell

Article Title: SIK2 is a centrosome kinase required for bipolar mitotic spindle formation that provides a potential target for therapy in ovarian cancer

doi: 10.1016/j.ccr.2010.06.018

Figure Lengend Snippet: A) A flow diagram showing the design of the in vivo experiment. B) Immunohistochemical confirmation of SIK2 depletion in SKOv3ip1 tissue sections using anti-SIK2 antibody. Scale bar, 50 µm. C–F) 5 µm sections from tumors were stained using the indicated antibodies. C–D) sections were stained using the indicated antibodies to calculate the mitotic index per field by manually counting phospho-Histone H3 positive nuclei and total number of nuclei. Shown in C is the mean ± s.e.m. of the MIs from 7 fields from two mice. In E an example of monopolar centrosome orientation following SIK2 KD in mice is shown. F) Bar plot representation of the mean ± s.e.m. of the percentage of mitotics with monopolar centrosome orientation from two mice (two slides per mouse). G–H) Mean tumor weights (± s.d.) obtained from 10 mice per siRNA type in SKOv3 cells and Hey cells. Scale bar, 10 µm.

Article Snippet: However, in G2, SIK2 expression resulted in a greater magnitude of C-Nap1 displacement (14.5% in SIK2 negative cells to 29.4% in SIK2 positive cells; p=0.004; Chi square test, n= 340, ).

Techniques: In Vivo, Immunohistochemical staining, Staining