Journal: Cell Death & Disease

Article Title: CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation

doi: 10.1038/s41419-021-04335-x

Figure Lengend Snippet: A Schematic outlining the proteomics experimental design and workflow to identify CaMKK2-interacting proteins by co-immunoprecipitation (co-IP). HEK293 cells were transfected with EGFP-tagged CaMKK2, co-immunoprecipitated proteins were identified by mass spectrometry and the data were compared with a previously published data set . B Immunoprecipitation of endogenous CaMKK2 from LNCaP cells showed co-immunoprecipitation of the Gemin complex. Immunoprecipitates (IP) with a control IgG were compared with those of CaMKK2 IgG, input lysate and unbound supernatant (SN). Bound proteins were evaluated by western blotting with the indicated antibodies. C A peptide array of full-length Gemin4 divided into 20 amino acid peptides with a 17 amino acid overlap was spotted on two nitrocellulose membranes. The membranes were incubated with either GST-CaMKK2 or GST alone. Bound GST protein was detected with a HRP-conjugated GST antibody. Yellow circles indicate peptides that bound CaMKK2 and had overlapping sequence with neighbouring peptides and red circles denote peptides that bound CaMKK2 but did not contain sequence overlap. A schematic drawing illustrates the sequence location of the highlighted peptides, which were concentrated in the first 370 aa of the Gemin4 sequence. D A peptide array of the N-terminal region of Gemin4 (aa 1–370) using 20 aa peptides with a 19 aa overlap. The peptide array was incubated with GST-CaMKK2 and GST control. The yellow box highlights the region in Gemin4 where GST-CaMKK2 but not GST bound. E Multiple sequence alignment of the CaMKK2-binding motif (CBM; aa 351–359) in Gemin4 identified in the peptide array illustrates sequence conservation across species. F To verify the specificity of the interaction in solution recombinant GST-tagged Gemin4 (aa 1–370) was used in a pulldown assay with LNCaP cell extracts. Equal amounts of control GST and GST-Gemin4 were used in the assay. The CaMKK2 binding was detected by western blotting.

Article Snippet: The short hairpin RNA (shRNA) constructs for CAMKK2 were obtained from the NKI library in pRETROSUPER vector: shControl (5’-ATTACTGCCTTTGGCCTCG-3’), shCAMKK2-1 (5’- CTTAAGAGACAACTAAGCC-3’), and shCAMKK2-2 (5’GGGCTTGAAATTTAATAAG-3’); and the pEGFP-C-shRNA constructs for GEMIN4 from Origene (Cat# TL304365V, Origene).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Mass Spectrometry, Control, Western Blot, Peptide Microarray, Incubation, Sequencing, Binding Assay, Recombinant

Journal: Cell Death & Disease

Article Title: CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation

doi: 10.1038/s41419-021-04335-x

Figure Lengend Snippet: A A schematic outlining the Gemin4 peptide pulldown experiment using biotinylated CaMKK2-binding motif (CBM) as bait from cell lysates of LNCaP cells treated with androgen (R1881) or an ethanol control. Bound proteins were identified by western blotting or mass spectrometry. B The streptavidin pulldown of biotinylated Gemin4 CBM and control peptide was analysed by western blotting to demonstrate enrichment of known interactors CaMKK2 and Gemin proteins. C Proteins that bound specifically to the Gemin4 CBM peptide were identified with mass spectrometry and the data set was assessed by pathway analysis (GO cellular location). The top ranked pathways are shown in a table. D A Gemin4 CBM peptide pulldown was used to validate the novel interactions of Gemin4 identified by the pathway analysis. Proteins bound by the peptide from LNCaP cell extract was analysed by western blot using antibodies for CaMKK2 and three components of the COPI complex; α-COP, δ-COP and ζ-COP.

Article Snippet: The short hairpin RNA (shRNA) constructs for CAMKK2 were obtained from the NKI library in pRETROSUPER vector: shControl (5’-ATTACTGCCTTTGGCCTCG-3’), shCAMKK2-1 (5’- CTTAAGAGACAACTAAGCC-3’), and shCAMKK2-2 (5’GGGCTTGAAATTTAATAAG-3’); and the pEGFP-C-shRNA constructs for GEMIN4 from Origene (Cat# TL304365V, Origene).

Techniques: Binding Assay, Control, Western Blot, Mass Spectrometry

Journal: Cell Death & Disease

Article Title: CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation

doi: 10.1038/s41419-021-04335-x

Figure Lengend Snippet: A Representative western blot of cell lysates from two CaMKK2 knockdown LNCaP cell lines. The efficiency of knockdown was quantified relative to GAPDH loading control. Mean ± SD; n = 3; * P = 0.0140; *** P = 0.0006. B Proliferation assay of LNCaP CaMKK2 KD in steady state conditions at 24, 48 and 72 h. Mean ± SD; n = 3; * P < 0.05; ** P < 0.01; *** P < 0.001. C Percentage of Annexin V and PI-positive cells present in LNCaP KD following 72 h in 10% foetal calf serum (FCS) or 10% charcoal-stripped serum (CSS) serum assessed by flow cytometry. Mean ± SD; N = 3; * P < 0.0170, ** P < 0.006, *** P < 0.0008. D Representative western blots of steady-state level COPI and Gemin4 protein levels in CaMKK2 knockdown (shRNA2) LNCaP cells. Densitometry values for δ-COP were generated in ImageJ relative to loading control. N = 3 for shRNA1 and N = 4 for shRNA2; P = 0.0281 for CAMKK2 shRNA1 and *** P = 0.0009 for CAMKK2 shRNA2 compared to control. E , F Transmission electron microscopic (TEM) images from CAMKK2 knockdown (shRNA2) and control LNCaP cells grown in regular media showing morphological differences in Golgi network (arrows), lipid droplets (LD) and autophagic vesicles (AV). N denotes nucleus. Scale bars 2 µm. G , H Electron micrographs of control and CaMKK2-depleted LNCaP cells showing the Golgi apparatus at higher magnification. Clathrin-coated membrane buds are marked in orange and non-clathrin-coated vesicles, presumably COPI coated, are marked in cyan. Inset shows COP-coated membrane buds; scale bar 50 nm. Scale bars 2 µm. I Quantification of vesicles per length of cis -Golgi membrane. Mean ± SD; n = 22; **** P < 0.0001. J Immunostaining of TGN46 (Golgi) in magenta showed a more dispersed distribution in the LNCaP CAMKK2 shRNA2 cell line compared to control. Endogenous calnexin (green) shows the endoplasmic reticulum. Scale bar = 10 µm. Boxed inlays are shown below. Scale bar = 5 µm. K Representative images showing maximal projections from z -stack confocal series of TGN46 (magenta) and DAPI (blue). L Quantification of the Golgi area normalised to the whole-cell area show an increase in Golgi area in CAMKK2-deficient cells. Sixty-two cells were quantified across three replicates for each cell line. Mean ± SD; *** P = 0.0004.

Article Snippet: The short hairpin RNA (shRNA) constructs for CAMKK2 were obtained from the NKI library in pRETROSUPER vector: shControl (5’-ATTACTGCCTTTGGCCTCG-3’), shCAMKK2-1 (5’- CTTAAGAGACAACTAAGCC-3’), and shCAMKK2-2 (5’GGGCTTGAAATTTAATAAG-3’); and the pEGFP-C-shRNA constructs for GEMIN4 from Origene (Cat# TL304365V, Origene).

Techniques: Western Blot, Knockdown, Control, Proliferation Assay, Flow Cytometry, Generated, Transmission Assay, Membrane, Immunostaining

Journal: Cell Death & Disease

Article Title: CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation

doi: 10.1038/s41419-021-04335-x

Figure Lengend Snippet: A LNCaP cells were treated with shRNA targeting GEMIN4 for 48 h. Protein expression levels were assessed by western blotting. Actin was used as loading control. B LNCaP cells were transiently transfected with a Gemin4 expression construct and protein expression levels were assessed by western blotting. Actin was used as a loading control. C Cell proliferation in Gemin4 knockdown and overexpressing LNCaP cells was compared to the respective control: control shRNA and EGFP. Cells were grown for 48 h in media supplemented with 10% charcoal-stripped serum. Mean ± SD, N = 3 independent experiments with 3 technical repeats; ** P = 0.0064, ns = 0.72. D Quantification of Golgi area (TGN46-postive) from confocal images (shown in E , F ) in Gemin4 knockdown and overexpressing LNCaP cells. Mean ± SD. Twenty-two knockdown cells and 20 overexpressing cells were analysed per group. ** P = 0.0030, ns = 0.24. E , F Images showing maximum intensity z -projections of confocal images of LNCaP cells transiently expressing EGFP-tagged shRNA or a Gemin4 expression constructs. Cell were stained with a TGN46 antibody (magenta). Scale bar, 25 µm. G Bar graph showing proliferation of LNCaP cells treated with 25 µM STO-609 for 120 h normalised to the DMSO control. Three independent experiments with three technical repeats were analysed. Mean ± SD; *** P = 0.0002. H LNCaP cells were treated with 25 µM STO-609 for 120 h. Protein expression of COPI components and Gemins were assessed by western blotting of 25 µg cell extracts separated on a SDS-PAGE gel. Actin was used as a loading control. Densitometry was performed on two experiments and normalised to control. Mean ± SD. I Bar graph showing the impact of 25 µM STO-609 treatment for 120 h in LNCaP cells on Golgi area (TGN46-positive) compared to CaMKK2 knockdown (shRNA2) cells. Mean ± SD. N = 31 cells; **** P < 0.0001.

Article Snippet: The short hairpin RNA (shRNA) constructs for CAMKK2 were obtained from the NKI library in pRETROSUPER vector: shControl (5’-ATTACTGCCTTTGGCCTCG-3’), shCAMKK2-1 (5’- CTTAAGAGACAACTAAGCC-3’), and shCAMKK2-2 (5’GGGCTTGAAATTTAATAAG-3’); and the pEGFP-C-shRNA constructs for GEMIN4 from Origene (Cat# TL304365V, Origene).

Techniques: shRNA, Expressing, Western Blot, Control, Transfection, Construct, Knockdown, Staining, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Gemin2 Plays an Important Role in Stabilizing the Survival of Motor Neuron Complex

doi: 10.1074/jbc.m609297200

Figure Lengend Snippet: FIGURE 1. Identification of a novel protein-protein interaction; Gemin2 self-interaction. A, gel-filtration analysis of Gemin2. The chromatogram demon- strates the elution of purified recombinant Gemin2 (*, the major elution peak of the protein). The arrows indicate the elution point of each molecular standard protein, and numbers correspond to the proteins shown at the inset graph. The inset graph indicates the semi-log plots for the molecular mass of the standard proteins and Gemin2 against the elution volume of these proteins in gel-filtration chromatography. B, the result of the mammalian two-hybrid assay. This experiment was independently conducted three times, and the errors bars represent standard deviation. Gal4-Gemin2 and VP16-SMN, Gemin2, and Gemin4 were expressed in CHO-K1 cells. The combination of Gal4-Gemin2 and VP16-Gemin4 was examined as a negative control. Protein-protein interaction was determined by measurement of luciferase reporter activity. C, the result of in vitro pull-down assay. In vitro translated 35S-labeled Gemin2 was incubated with in vitro translated biotinylated SMN, Gemin2, and Luc (luciferase as a negative control) and formed complexes that were captured with streptavidin beads. Proteins that remained bound to the beads were analyzed by SDS-PAGE and visualized by autoradiography. 10% of 35S-labeled protein used in the assay was loaded as an input. D, Gemin2 self-association using the purified recombinant Gemin2. Purified GST, GST-Gemin2, and Gemin2-His were resolved by SDS-PAGE and visualized by Coomassie staining (left panel). Right panels show the results of the GST pull-down assay. Purified GST or GST-Gemin2 were immobilized on glutathione-Sepharose and incubated with purified Gemin2-His. After resolution by SDS-PAGE, the bound proteins were visualized by Coomassie staining or Western blot using an anti-Gemin2 or an anti-His antibody. 30% of the Gemin2-His used in each reaction were loaded as an input.

Article Snippet: Aswell asGemin2, the roles and themechanisms of the other Gemin proteins in the SMN complex are not yet fully characterized, although recent analysis reveals that, in addition to the SMN protein, Gemin3, -5, -6, and -7 all associate directly with the Sm proteins (24), and Gemin2, -3, -4, -6, and -8 and SMN are important for the U1 snRNP assembly activity of the SMN complex (20, 25, 26).

Techniques: Filtration, Purification, Recombinant, Chromatography, Two Hybrid Assay, Standard Deviation, Negative Control, Luciferase, Activity Assay, In Vitro, Pull Down Assay, Labeling, Incubation, SDS Page, Autoradiography, Staining, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Gemin2 Plays an Important Role in Stabilizing the Survival of Motor Neuron Complex

doi: 10.1074/jbc.m609297200

Figure Lengend Snippet: FIGURE 2. Interaction domain mapping for Gemin2 self-association and Gemin2-SMN association. A, schematic representation of the full-length and deletion mutants of Gemin2 (left panel). The interaction was examined using a mammalian two-hybrid assay. Gal4-fused proteins for full-length Gemin2 or Gemin2-deletion mutants were expressed in CHO-K1 cells with VP16-SMN (middle panel) or VP16-Gemin2 or VP16-Gemin2 mutants (right panel). Protein- protein interactions were determined with the same procedures as in Fig. 1B. The experiment was independently conducted three times, and the errors bars represent the standard deviation. B, confirmation of the properties of the Gemin2 self-association and the Gemin2-SMN association in the mutant Gemin290 –269. The interaction assay was performed by the same method as in Fig. 1C.

Article Snippet: Aswell asGemin2, the roles and themechanisms of the other Gemin proteins in the SMN complex are not yet fully characterized, although recent analysis reveals that, in addition to the SMN protein, Gemin3, -5, -6, and -7 all associate directly with the Sm proteins (24), and Gemin2, -3, -4, -6, and -8 and SMN are important for the U1 snRNP assembly activity of the SMN complex (20, 25, 26).

Techniques: Two Hybrid Assay, Protein-Protein interactions, Standard Deviation, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Gemin2 Plays an Important Role in Stabilizing the Survival of Motor Neuron Complex

doi: 10.1074/jbc.m609297200

Figure Lengend Snippet: FIGURE 5. Comparison of SMN oligomerization between untreated HeLa cells and Gemin2 knockdown cells. A, evaluation of gene expression of SMN complex components in HeLa cells following Gemin2 knock- down. The relative expression levels of the target genes were determined by qRT-PCR using total RNA from cells 44 h after transfection with Gemin2 siRNA. The black and white bars show the results of qRT-PCR analysis using two different primer sets against each target gene, and the errors bars represent the standard deviation. B, Gemin2 gene silencing with siRNA. Whole cell extracts were prepared from untreated, Gemin2 siRNA-trans- fected (siGemin2), negative control siRNA transfected (siControl), and mock transfected (Mock) HeLa cells. The cell extracts were analyzed by Western blotting using anti-SMN, anti-Gemin2, and anti-PRMT5 polyclonal antibodies. PRMT5 was used as loading control. C, sedimentation analysis of the cell extracts of native HeLa cells (WT, top panel), Gemin2 knockdown cells with siRNA (siGemin2, middle panel), and cells transfected with negative control siRNA (siControl, bottom panel). The cell extracts were sedimented on a 6–38% (w/v) sucrose density gradient. Fractions, indicated by numbers 1–13 from top to bottom of the centrifuge tube, were sub- jected to SDS-PAGE followed by Western blotting using anti-SMN polyclonal antibody. D, cytoplasmic soluble fraction from the siRNAs (siGemin2 or siControl) treated and untreated (WT) HeLa cells were immunoprecipi- tated with anti-SMN antibody. 2% of the input (left panel) and immunoprecipitates (right panel) were analyzed by Western blotting using anti-SMN, Gemin2, Gemin3, Gemin7, and SmB/B antibodies.

Article Snippet: Aswell asGemin2, the roles and themechanisms of the other Gemin proteins in the SMN complex are not yet fully characterized, although recent analysis reveals that, in addition to the SMN protein, Gemin3, -5, -6, and -7 all associate directly with the Sm proteins (24), and Gemin2, -3, -4, -6, and -8 and SMN are important for the U1 snRNP assembly activity of the SMN complex (20, 25, 26).

Techniques: Comparison, Knockdown, Gene Expression, Expressing, Quantitative RT-PCR, Transfection, Standard Deviation, Negative Control, Western Blot, Control, Sedimentation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Gemin2 Plays an Important Role in Stabilizing the Survival of Motor Neuron Complex

doi: 10.1074/jbc.m609297200

Figure Lengend Snippet: FIGURE 6. The SMA-derived mutant SMN(D44V) caused a reduction in amino-terminal self-association and Gemin2 binding. Assay samples for the mutant SMNs were constructed from wild-type human SMN cDNA and were subjected to in vivo (A and B) and in vitro (C and D) binding assays as described above. The in vivo binding assay was independently conducted four times, and the errors bars represent standard deviation. The lower numerical values (C and D) indicate the percentage of the pull-downed 35S-labeled proteins against 10% of 35S-labeled protein used in the assay.

Article Snippet: Aswell asGemin2, the roles and themechanisms of the other Gemin proteins in the SMN complex are not yet fully characterized, although recent analysis reveals that, in addition to the SMN protein, Gemin3, -5, -6, and -7 all associate directly with the Sm proteins (24), and Gemin2, -3, -4, -6, and -8 and SMN are important for the U1 snRNP assembly activity of the SMN complex (20, 25, 26).

Techniques: Derivative Assay, Mutagenesis, Binding Assay, Construct, In Vivo, In Vitro, Standard Deviation, Labeling