Journal: Theranostics

Article Title: IKKε phosphorylates kindlin-2 to induce invadopodia formation and promote colorectal cancer metastasis

doi: 10.7150/thno.40397

Figure Lengend Snippet: IKKε directly phosphorylates kindlin-2 at S159. (A) Slot blot analysis of the anti-phospho-kindlin-2-S159 antibody. The antibody was probed against an un-modified kindlin-2 peptide and phosphorylated peptides. (B) HCT116 cells were transfected with the indicated plasmids and whole cell extracts were immunoprecipitated (IP) with FLAG-conjugated M2 beads and then analyzed by immunoblotting (IB). (C)HCT116 cells were treated with the inhibitor IKK-3 at the indicated concentration for 24h and analyzed by immunoblotting. (D and E) HCT116 cells were transfected with the indicated siRNAs targeting IKKα, IKKβ, IKKε or TBK1. Cell lysates were analyzed by immunoblotting. (F) HCT116 cells were transfected with FLAG-tagged kindlin-2 and GFP-tagged IKKε for 48 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. (G) IKKε was immunoprecipitated using anti-IKKε antibody or the control antibody IgG from lysates of HCT116 cells. (H) HCT116 cells were transfected with the indicated plasmids and the whole cell extracts were prepared and analyzed by Co-IP assay flowed by immunoblotting with the indicated antibodies. (I) GST-tagged wild-type kindlin-2 or kindlin-2 (S159A) were incubated without IKKε (-), or with purified wild-type IKKε (WT) or mutant IKKε (K38A) in kinase buffer and resolved by SDS-PAGE, followed by immunoblotting analysis with the indicated antibodies.

Article Snippet: Co. Ltd. Antibodies against IKKε (ab7891, Abcam) and phospho-kindlin-2 (S159) (CTM-152, PTM Biolabs) were used for immunohistochemistry staining according to the manufacture's protocol.

Techniques: Dot Blot, Modification, Transfection, Immunoprecipitation, Western Blot, Concentration Assay, Co-Immunoprecipitation Assay, Incubation, Purification, Mutagenesis, SDS Page

Journal: Theranostics

Article Title: IKKε phosphorylates kindlin-2 to induce invadopodia formation and promote colorectal cancer metastasis

doi: 10.7150/thno.40397

Figure Lengend Snippet: IKKε-induced invadopodia formation depends on kindlin-2 S159 phosphorylation. (A) HCT116 cells were transfected with GFP-tagged IKKε and stained with antibodies against kindlin-2, Tks5 and F-actin (phalloidin). Confocal images were acquired to visualize the localization of IKKε, kindlin-2, Tks5 and F-actin. White arrowhead point to the co-localization of IKKε, kindlin-2, Tks5 and F-actin. Scale bar, 10 μm. (B) Co-localization of Tks5 and F-actin in HCT116 cells with kindlin-2 knockout and rescue with wild-type IKKε, wild-type kindlin-2, phosphor-null mutant kindlin-2 (S159A) and phosphor-mimetic mutant (S159D) kindlin-2 (left). Inset shows magnified image of invadopodia in the box. Quantification of cells with invadopodia (right). (C) Gelatin degradation in HCT116 cells with kindlin-2 knockout and rescue with wild-type IKKε, wild-type kindlin-2, phosphor-null mutant kindlin-2 (S159A) and phosphor-mimetic mutant (S159D) kindlin-2 (left). The area of degraded gelatin per cell (right). All data represent the means ± S.D. (N>400 cells/sample, * P <0.05, ** P < 0.01, and *** P < 0.001).

Article Snippet: Co. Ltd. Antibodies against IKKε (ab7891, Abcam) and phospho-kindlin-2 (S159) (CTM-152, PTM Biolabs) were used for immunohistochemistry staining according to the manufacture's protocol.

Techniques: Transfection, Staining, Knock-Out, Mutagenesis

Journal: Theranostics

Article Title: IKKε phosphorylates kindlin-2 to induce invadopodia formation and promote colorectal cancer metastasis

doi: 10.7150/thno.40397

Figure Lengend Snippet: Phosphorylated kindlin-2 (S159) levels positively correlate with IKKε expression in colorectal cancer tissues. (A) Immunohistochemical staining for phosphor-kindlin-2-S159 in human colorectal cancer tissues and paired adjacent normal tissues. Scale bar, 200 μm. (B) Phosphor-kindlin-2-S159 protein levels in 161 paired human colorectal cancer tissues and adjacent normal tissues as measured by immunohistochemical analysis. The data represent the means ± S.D. (*** P < 0.001). (C) Representative images of paraffin tissue sections stained with IKKε and phosphorylated kindlin-2 (S159) antibody. Scale bar, 200 μm. (D) Correlation analyses of IKKε and phosphorylated kindin-2 (S159) protein levels in human colorectal cancer specimens.

Article Snippet: Co. Ltd. Antibodies against IKKε (ab7891, Abcam) and phospho-kindlin-2 (S159) (CTM-152, PTM Biolabs) were used for immunohistochemistry staining according to the manufacture's protocol.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Genomics, Proteomics & Bioinformatics

Article Title: LDHA Desuccinylase Sirtuin 5 as A Novel Cancer Metastatic Stimulator in Aggressive Prostate Cancer

doi: 10.1016/j.gpb.2022.02.004

Figure Lengend Snippet: LDHA desuccinylation by SIRT5 is related to aggressive PCa A. LDHA activity was increased in SIRT5-depleted PCa in PC-3M, and PC-3 SIRT5-KO cells. LDHA activity was determined in PC-3 (Normal), Mock treated PC-3 (Mock), PC-3 SIRT5-KO (SIRT5 KO), and PC-3M cells. B. SIRT5 inhibition leaded to increased by LDHA activity. LDHA activity was determined by the treatment with the SIRT5 selective inhibitor (HY-112634) in PC-3 cells. C. Inducing succinylation increased LDHA activity. Succinate was administered to PC-3 cells to increase the succinylation level, and LDHA activity was determined. D. Detected succinylation sites (su) in LDHA. Among the detected sites, sites with succinylation levels increased more than two times in PC-3M are marked with circles. The acetylation (ac) and ubiquitylation (ub) sites known in phosphostie.org are indicated. E. LDHA with lysine (K) mutated to glutamic acid (E) at K118 residue increased LDHA activity. The four lysine residues K5, K76, K118, and K232, with up-regulated succinylation in PC-3M, were mutated to E to mimic the negatively charged succinyl-lysine modification. After PC-3 cells were transfected with LDHA-mutants using lipofectamine 3000, LDHA activities were determined. GAPDH served as a loading control ( n = 3). F. The mutation from K to E of LDHA at K118 promotes cell migration and invasion. Cell migration and invasion of PC-3 cells expressed LDHA-K118E were analyzed by wound-healing and transwell invasion assays ( n = 3). Representative images depict invading cells (200 ×). Cells that invaded through matrigel were stained with crystal violet. Bar, 25 mm. Data are presented as mean ± SE. Statistical analysis was performed using one-way analysis of variance and presented as follows: *, P < 0.05; **, P < 0.01 vs. mock; # , P < 0.05; ## , P < 0.01 vs. LDHA-WT. See also Figures S5 and S6. LDHA, lactate dehydrogenase A; WT, wild type.

Article Snippet: The polyclonal antibody specific for succinylated LDHA at K118 (anti-succinylated K118-LDHA, Catalog No. CTM-121) was produced in collaboration with PTM biolabs (Hangzhou, China).

Techniques: Activity Assay, Inhibition, Residue, Modification, Transfection, Control, Mutagenesis, Migration, Staining

Journal: Genomics, Proteomics & Bioinformatics

Article Title: LDHA Desuccinylase Sirtuin 5 as A Novel Cancer Metastatic Stimulator in Aggressive Prostate Cancer

doi: 10.1016/j.gpb.2022.02.004

Figure Lengend Snippet: LDHA succinylation promotes PCa progression A. LDHA acetylation induces LDHA degradation. Succinate increased succinylation at the K118 residue of LDHA. The levels of LDHA and LDHA-K118su were determined by Western blotting after treatment with acetate or succinate in PC-3 cells for 8 h. β-actin served as a loading control. B. The protein expression level of LDHA, LDHA-K118su, and SIRT5 in seven types of PCa cell lines was evaluated by immunoblot. β-actin served as a loading control. C. SIRT5 interacts with LDHA. The immunoblots showed the IP of SIRT5 with anti-LDHA antibodies in PC-3 cells and IP of LDHA with anti-SIRT5 antibodies in PC-3 cells, respectively. D. As PCa progresses, LDHA is succinylated at K118 becomes the K118su residue, along with increased LDHA activity. LDHA activity was determined in PCa tissues classified by pathological grade (BPH, T2, and T3 grades). The K118su level of LDHA in PCa tissue was measured by ELISA using the LDHA-K118su antibody. E. Working model of LDHA succinylation at K118 in aggressive PCa cells. LDHA degradation is regulated according to acetylation at the K5 position by SIRT2, according to Zhao et al. 2013 . In PCa, the SIRT5 protein level is reduced; The succinylation at K118 of LDHA as the substrate of SIRT5 is maintained. Succinylated LDHA is more active than the native LDHA, which can increase lactate production, ultimately leading to PCa progression. Data are presented as mean ± SE. Statistical analysis was performed using one-way analysis of variance and presented as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs . BPH. K118su, K118 succinylation.

Article Snippet: The polyclonal antibody specific for succinylated LDHA at K118 (anti-succinylated K118-LDHA, Catalog No. CTM-121) was produced in collaboration with PTM biolabs (Hangzhou, China).

Techniques: Residue, Western Blot, Control, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Bone Differentiation Ability of CD146-Positive Stem Cells from Human Exfoliated Deciduous Teeth

doi: 10.3390/ijms24044048

Figure Lengend Snippet: Surface-antigen analysis of SHED and comparative properties of SHED, CD146 + SHED, and CD146-SHED cellular proliferative potential. ( a ) Surface-antigen analysis of SHED. Among the heterogeneous population of SHED isolated from the deciduous pulp, 70.9 ± 4.3% of cells expressed CD146. MSCs were positive for CD90, CD73, and CD105 but negative for CD14, CD19, CD34, and CD45. ( b ) Comparison of population doubling time (PDT). The log phase was assessed based on the cell growth curve from day 2 to day 6 of incubation, and the PDT was calculated for this period. PDT did not differ significantly among the three groups ( n = 5, Kruskal–Wallis test, Not significant; N.S.) ( c , d ). Comparison of cell proliferation. ( c ) Two hours after BrdU treatment, DNA synthesized in SHED was slightly higher than that in CD146 + SHED and CD146-SHED, but there were no significant differences among the three groups. ( d ) Twenty-four hours after BrdU treatment, the results were similar ( n = 5, Kruskal–Wallis method, Not significant; N.S.).

Article Snippet: One of the prepared solutions was supplemented with 30 μL of PE mouse anti-human CD146, PE mouse anti-human CD90, FITC mouse anti-human CD105, PE-CTM 7 mouse anti-human CD14, APC-H7 mouse anti-human CD19 (BD Pharmingen, San Jose, CA, USA), Brilliant Violet™ 421 mouse anti-human CD73, FITC mouse anti-human CD34, and APC-H7 mouse anti-human CD45 (Becton Dickinson, San Jose, CA, USA).

Techniques: Isolation, Incubation, Synthesized

Journal: Experimental & Translational Stroke Medicine

Article Title: Coadministration of FTY720 and rt-PA in an experimental model of large hemispheric stroke–no influence on functional outcome and blood–brain barrier disruption

doi: 10.1186/2040-7378-5-11

Figure Lengend Snippet: FTY720 leads to a rapid decrease of blood lymphocyte counts. We sampled blood 2 h after i.p. injection of FTY720 (1 mg/kg). FACS analysis showed a significant reduction of both CD4+ and to a lesser extent CD8+ T cells (n = 3/group). Statistical significance between groups was tested with two-tailed Student’s t -test for unpaired values. * p < 0.05.

Article Snippet: After washing with RPMI, samples were incubated with FACS buffer (1% FCS in PBS/0,01% NaN3) and the respective antibodies (Rat anti-mouse CD4 [APC-coupled, Southern Biotech, 1540–11], rat anti-mouse CD8 [PerCP-CTM5.5-coupled, BD Pharmingen, 551162]) for 30 minutes before overnight fixation in 4% PFA in PBS at 4°C.

Techniques: Injection, Two Tailed Test