Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel cultivation strategy to recover NK cell cytotoxicity

doi: 10.3389/fbioe.2026.1797129

Figure Lengend Snippet: Total VCC, viability (A) , cytotoxicity, specific growth rate (B) , cytotoxic capacity (C) , lactate concentration and production rate (D) , glucose concentration and consumption rate (E) , glutamine concentration and consumption rate (F) of the NK-92 cell batch expansion process in static T-flasks. Error bars represent the standard deviation of three, independent biological replicates (n = 3).

Article Snippet: To assess the cytotoxicity of the NK-92 cells, K-562-GFP (K562) cells (CCL-243-GFP, American Type Culture Collection, Manassas, VA, United States) were maintained at standard cultivation conditions in a Roswell Park Memorial Institute 1640 medium (RPMI; Gibco, Grand Island, NY, United States) containing 5% heat-inactivated fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, United States).

Techniques: Concentration Assay, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel cultivation strategy to recover NK cell cytotoxicity

doi: 10.3389/fbioe.2026.1797129

Figure Lengend Snippet: (A) Graphical overview of the design space used to determine the cytotoxicity recovery without an additional factor of recovery duration. Overview of significant factors used for the calculation of the PLS model for cytotoxicity (B) and cytotoxic capacity (C) after normalization of the coefficients. Only significant factors are displayed (different than zero) with a standard deviation smaller than the factor’s value - Med: medium composition, Temp: cultivation temperature Dur: recovery duration. (D,E) Contour plots illustrating PLS-based predictions of the optimal recovery setpoint, derived from the analysis of DoE responses for cytotoxicity and cytotoxic cells. The circles on both plots represent the combined optimum conditions that maximize cytotoxicity and cytotoxic capacity recovery. The factor recovery duration was set to the identified optimum of 3.7 days.

Article Snippet: To assess the cytotoxicity of the NK-92 cells, K-562-GFP (K562) cells (CCL-243-GFP, American Type Culture Collection, Manassas, VA, United States) were maintained at standard cultivation conditions in a Roswell Park Memorial Institute 1640 medium (RPMI; Gibco, Grand Island, NY, United States) containing 5% heat-inactivated fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, United States).

Techniques: Standard Deviation, Derivative Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel cultivation strategy to recover NK cell cytotoxicity

doi: 10.3389/fbioe.2026.1797129

Figure Lengend Snippet: Time-resolved measurements of cytotoxicity (A) and cytotoxic capacity (B) during the cytotoxicity recovery process. Cytotoxicity (C) , cytotoxic capacity (D) , total viable cell count (E) , viability (F) , cumulative population doublings (G) , and STY (H) were compared between medium compositions on day 3 when cytotoxicity recovery reached its maximum, using one-way ANOVA, where p ≥ 0.05 is not significant (ns), p < 0.05 is *, p < 0.01 is **, p < 0.001 is ***, and p < 0.0001 is ****. Error bars represent standard deviations of all runs per medium composition (n = 4 for fresh medium (green), n = 8 for mixed medium (red) and n = 4 for spent medium (blue)). Correlation between cytotoxicity at day 3 of the recovery and lactate concentration (I) and pH (J) at day 0 of cytotoxicity recovery. Regression was calculated with simple linear regression and correlation coefficients were calculated with Pearson’s correlation.

Article Snippet: To assess the cytotoxicity of the NK-92 cells, K-562-GFP (K562) cells (CCL-243-GFP, American Type Culture Collection, Manassas, VA, United States) were maintained at standard cultivation conditions in a Roswell Park Memorial Institute 1640 medium (RPMI; Gibco, Grand Island, NY, United States) containing 5% heat-inactivated fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, United States).

Techniques: Cell Characterization, Concentration Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A novel cultivation strategy to recover NK cell cytotoxicity

doi: 10.3389/fbioe.2026.1797129

Figure Lengend Snippet: Total viable cell count and viability (A) , cytotoxicity and lactate concentration (C) , and cytotoxic capacity and specific growth rate (E) of NK-92 cells during a fed-batch expansion process in a 2-L working volume stirred-tank bioreactor. Total VCC and viability (B) , cytotoxicity and lactate concentration (D) , and cytotoxic capacity and specific growth rate (F) of NK-92 cells during the cytotoxicity recovery phase following expansion in the same bioreactor are shown. Due to the limited working volume of the bioreactor, only 6.6% of all cells produced during the fed-batch phase could be retained for the recovery phase (hence, different absolute values of the total viable cell count and cytotoxic capacity are displayed). Viablity at harvest (G) and STY at harvest (H) after the expansion process phase and the recovery process phase were compared uising an unpaired t-test where p ≥ 0.05 is not significant (ns), p < 0.05 is *, p < 0.01 is **, p < 0.001 is ***, and p < 0.0001 is ****. Error bars for expansion process and recovery process represent technical triplicates (n = 3).

Article Snippet: To assess the cytotoxicity of the NK-92 cells, K-562-GFP (K562) cells (CCL-243-GFP, American Type Culture Collection, Manassas, VA, United States) were maintained at standard cultivation conditions in a Roswell Park Memorial Institute 1640 medium (RPMI; Gibco, Grand Island, NY, United States) containing 5% heat-inactivated fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, United States).

Techniques: Cell Characterization, Concentration Assay, Produced

Journal: Pharmaceutics

Article Title: Modulation of the Cytotoxic Properties of Pd(II) Complexes Based on Functionalized Carboxamides Featuring Labile Phosphoryl Coordination Sites

doi: 10.3390/pharmaceutics15041088

Figure Lengend Snippet: Cytotoxicity of the phosphoryl-functionalized amide derivatives against some human hematopoietic c ancer cell lines.

Article Snippet: To study the apoptosis inducing ability of complex 3b , K562 and K562/iS9 cells, preincubated for a day in a CO 2 incubator at 37 °C, were cultured in the medium containing 10 μM of the palladocycle for 20 h. After exposure, the cells were washed with cold PBS and incubated with Annexin V-FITC for 20 min before being treated with PI according to the supplier protocol (Elabscience Annexin V-FITC/PI Apoptosis Detection Kit).

Techniques:

Journal: Pharmaceutics

Article Title: Modulation of the Cytotoxic Properties of Pd(II) Complexes Based on Functionalized Carboxamides Featuring Labile Phosphoryl Coordination Sites

doi: 10.3390/pharmaceutics15041088

Figure Lengend Snippet: Percentages of necrotic (upper left), early apoptotic (lower right), and late apoptotic (upper right) K562 ( a , b ) and K562/iS9 ( c , d ) cells in the control experiments ( a , c ) and after exposure to complex 3b ( b , d ) for 20 h.

Article Snippet: To study the apoptosis inducing ability of complex 3b , K562 and K562/iS9 cells, preincubated for a day in a CO 2 incubator at 37 °C, were cultured in the medium containing 10 μM of the palladocycle for 20 h. After exposure, the cells were washed with cold PBS and incubated with Annexin V-FITC for 20 min before being treated with PI according to the supplier protocol (Elabscience Annexin V-FITC/PI Apoptosis Detection Kit).

Techniques: Control

Journal: RMD Open

Article Title: Development of an enzyme-linked immunosorbent assay for the efficient detection of autoantibodies against nuclear valosin-containing protein-like protein (NVL) 2 using its manipulated cDNA

doi: 10.1136/rmdopen-2025-005679

Figure Lengend Snippet: Representative results of IP-WB using K562 nuclear extract for anti-NVL2 antibodies and an indirect immunofluorescence image for an anti-NVL2 antibody-positive serum sample. ( A ) Immunoprecipitates from K562 nuclear extracts with patient’s serum were probed with anti-NVL polyclonal antibody to confirm the ELISA results using the recombinant protein. Of the 28 ELISA high-titre sera, 18 sera showed reactivity with an approximately 95 kDa band by IP-WB. Lane 1 shows an input lane that contains half the dose of K562 extract used. Lanes 2, 3 and 4 correspond to Pt. 1, 7 and 11 in , respectively. Lanes 5 and 6 correspond to IP-WB negative patients among the ELISA high-titre sera. Lane 7 is serum from a healthy control. An approximately 95 kDa band was detected in lanes 2–4, but not in lanes 5–7. Lane 7 is serum from a healthy control. *denotes the position of 95 kDa molecular weight proteins in the IP-WB analysis. ( B ) In the AC-8 pattern seen by indirect immunofluorescence on anti-NVL2 antibody-positive serum sample, we see diffuse fluorescence of the entire nucleolus (blue arrowhead) and no staining of the metaphase plate (red arrowhead). IP, immunoprecipitation; NVL, nuclear valosin-containing protein-like protein; Pt, patient; WB, Western blotting

Article Snippet: IP-WB was performed using K562 nuclear cell extracts (Santa Cruz Biotechnology, Dallas, Texas, USA) without cross-linking, as described previously, with minor modifications.

Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay, Recombinant, Control, Molecular Weight, Fluorescence, Staining, Immunoprecipitation, Western Blot

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Schematic representation of human CENP-C protein. The Mis12-binding domain (M12BD) of human CENP-C is highlighted in wild-type (WT) CENP-C. Cells expressing CENP-C lacking M12BD (CENP-C ΔM12BD cells) were generated (see also ). (B) Schematic representation of the pooled CRISPR screening in K562 WT or CENP-C ΔM12BD cells. Using CRISPR viability scores, genes which are essential for cell growth in K562 CENP-C ΔM12BD cells but not in K562 WT, cells were selected as candidates whose knockout showed synthetic lethality with CENP-C ΔM12BD . (C) Scatterplots showing the CRISPR viability score in K562 WT versus CENP-C ΔM12BD cell pools. Red dots are essential genes in K562 CENP-C ΔM12BD cells but not in K562 WT cells. (D) Gene set enrichment analysis (GSEA) for 375 candidate genes. The candidate genes were ranked by the magnitude of differences in CRISPR viability score between K562 WT and CENP-C ΔM12BD cells in descending order and applied to GSEA. The top 10 enriched gene sets were highlighted. (E) GSEA result of GOCC_SPINDLE_POLE gene set. The heatmap indicates the CRISPR viability score of the leading-edge genes of the preranking based on the magnitude of differences in CRISPR viability score between K562 WT and CENP-C ΔM12BD cells, in descending order. (F) GSEA result of GOMF_TUBULIN_BINDING gene set. The heatmap shows as in (E).

Article Snippet: To localize CENP-C, CENP-T, CENP-A, DSN1, KNL1, HEC1, and MAD2 in K562 cells, K562 cells were cytospan onto slide glasses (MATSUNAMI, S2111) or coverslips (MATSUNAMI, C024361) and then fixed with 3% paraformaldehyde (PFA; Nacalai Tesque) for 10 min at RT, and rinsed with PBS.

Techniques: Binding Assay, Expressing, Generated, CRISPR, Knock-Out

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Cell viability of K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days after doxycycline (Dox) addition (mean and SD, two-tailed Student’s t test, CENP-C WT cells: n = 6; CENP-C ΔM12BD cells: n = 6; ** p < 0.01). (B) Representative images of DAPI-stained K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Scale bar, 50 μm. (C) Population of normal interphase cells, mitotic cells, and cells with micronuclei in K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Error bars indicate SEM. n = 3 independent experiments; 200 cells from each cell line were quantified in each experiment. (D) The growth curve of K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. K562 WT or CENP-C ΔM12BD cells were treated with or without Dox ( KIF18A OFF or ON). The cell numbers were normalized to those at time 0 of each line. (E) Cell-cycle distribution of conditional knockout of KIF18A in K562 WT cells at each day after Dox addition, based on FACS analysis. (F) Cell-cycle distribution of conditional knockout of KIF18A in K562 CENP-C ΔM12BD cells at each day after Dox addition, based on FACS analysis. (G and H) Quantification of cells with misaligned chromosomes in K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. The experimental scheme is shown. Cells were stained with antibodies against MAD2 (red) to detect misaligned chromosomes and CENP-T (green) as a kinetochore marker. DNA was stained with DAPI (blue). Arrowheads show typical MAD2-positive unaligned chromosomes. Scale bar, 10 μm. The cells with MAD2-positive chromosomes were quantified (H) (mean and SEM, two-tailed Student’s t test; n = 5 independent experiments; n.s., non-significant; ** p < 0.01). (I) Numbers of MAD2 positive kinetochores in each cell in each condition (WT KIF18A ON; WT KIF18A OFF; CENP-C ΔM12BD KIF18A ON; CENP-C ΔM12BD KIF18A OFF) (Mean and SEM, n = 5 independent experiments).

Article Snippet: To localize CENP-C, CENP-T, CENP-A, DSN1, KNL1, HEC1, and MAD2 in K562 cells, K562 cells were cytospan onto slide glasses (MATSUNAMI, S2111) or coverslips (MATSUNAMI, C024361) and then fixed with 3% paraformaldehyde (PFA; Nacalai Tesque) for 10 min at RT, and rinsed with PBS.

Techniques: Knock-Out, Two Tailed Test, Staining, Marker

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Cell viability of RPE-1, K562, U2OS, A549, TIG-3, HeLa, HT1080, OVCAR-3, HT29, and HCC1806 cells with or without KIF18A knockdown (mean and SD, two-tailed Student’s t test; each sample size: n = 6; ** p < 0.01). (B–D) CENP-E (B), CENP-C (C), and CENP-T (D) localization in RPE-1, K562, U2OS, A549, TIG-3, HeLa, HT1080, OVCAR-3, HT29, and HCC1806 cells. The cell lines were treated with 50 μM monastrol for 2 h to enrich prometaphase cells. Each cell line was cytospun together with monastrol-treated RPE-1 cells expressing mScarlet-CENP-A as an internal control for immunostaining. CENP-E (green) (B), CENP-C (green) (C), and CENP-T (green) (D) were stained with antibodies against each protein. DNA was stained with DAPI (blue). CENP-T or CENP-A was stained as a kinetochore marker (red). Scale bar, 10 μm. CENP-E, CENP-C, or CENP-T signal intensities at mitotic kinetochores were quantified and normalized with those of the internal control RPE-1 mScarlet-CENP-A cells in each sample (mean and SD, two-tailed Student’s t test, each cell line: n = 10 cells; n.s., non-significant; * p < 0.1; ** p < 0.01).

Article Snippet: To localize CENP-C, CENP-T, CENP-A, DSN1, KNL1, HEC1, and MAD2 in K562 cells, K562 cells were cytospan onto slide glasses (MATSUNAMI, S2111) or coverslips (MATSUNAMI, C024361) and then fixed with 3% paraformaldehyde (PFA; Nacalai Tesque) for 10 min at RT, and rinsed with PBS.

Techniques: Knockdown, Two Tailed Test, Expressing, Control, Immunostaining, Staining, Marker

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: In wild-type K562 and RPE-1 cells with KIF18A depletion (middle: + KIF18A KD), although KIF18A depletion enhances microtubule dynamics and increases peripheral chromosomes, CENP-E downstream of CENP-C compensates for KIF18A depletion, supporting chromosome congression and alignment, albeit with a delay in the congression. By contrast, in CENP-E-deficient cells (bottom: K562 CENP-C ΔM12BD and RPE-1 CENP-C ΔM12BD cells), KIF18A depletion disrupts chromosome congression due to weakened CENP-E activity downstream of CENP-C at kinetochores, leading to mitotic arrest and subsequent cell death. Based on these observations, we propose that the CENP-C pathway plays a role in chromosome congression, with KIF18A and CENP-E acting cooperatively to promote the congression of peripheral chromosomes during early prometaphase in wild-type K562 and RPE-1 cells (upper).

Article Snippet: To localize CENP-C, CENP-T, CENP-A, DSN1, KNL1, HEC1, and MAD2 in K562 cells, K562 cells were cytospan onto slide glasses (MATSUNAMI, S2111) or coverslips (MATSUNAMI, C024361) and then fixed with 3% paraformaldehyde (PFA; Nacalai Tesque) for 10 min at RT, and rinsed with PBS.

Techniques: Activity Assay