metastatic pdac cell lines capan 1  (ATCC)

Journal: bioRxiv

Article Title: IL12-engineered human PSMA-CAR T cells for the treatment of advanced prostate cancer

doi: 10.64898/2026.03.05.709907

Figure Lengend Snippet: Engineering hPSMA-CARs with mbIL12 rescues CAR T cell functionality a Schema of hPSMA-CAR variants with mbIL12 expression. b Flow cytometric analysis of ET260-1 CAR and ET260-1(58) CAR with or without mbIL12 as detected by extracellular Fc and intracellular IL-12. c Tumor cell killing of hPSMA-CARs at an E:T of 1:4 over 72 hrs. d Expression of 4-1BB over 72 hrs on hPSMA-CARs. e Expression of CD25 on hPSMA-CARs after 72 hrs. f Secretion of IFNγ of hPSMA-CARs with mbIL12 after 72 hrs as determined by ELISA. g Tumor cell killing of hPSMA-CARs with mbIL12 and J591 against PC-3 PIP at E:T of 1:10 over 5 days at varying concentrations of anti-IFNγR1 blocking antibody and isotype control. Data are presented as mean values ± SEM. P values indicate differences between ET260-1-dCH2(28tm)BBz and ET260-1-dCH2(28tm)BBz/mbIL12 and between ET260-1-dCH2(28tm)BBz/mbIL12 and J591-dCH2(4tm)BBz using a two-tailed Student’s t-test.

Article Snippet: Human metastatic prostate cancer cell line PC-3 (ATCC CRL-1435) was cultured in RPMI-1640 (Corning) containing 10% fetal bovine serum (FBS, Hyclone), and 1X antibiotic-antimycotic (AA, Gibco) (complete RPMI).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Blocking Assay, Control, Two Tailed Test

Journal: bioRxiv

Article Title: IL12-engineered human PSMA-CAR T cells for the treatment of advanced prostate cancer

doi: 10.64898/2026.03.05.709907

Figure Lengend Snippet: mbIL12-engineered hPSMA-CAR T cells demonstrate improved activity in a recursive tumor cell assay in vitro a Tumor cell killing of hPSMA-CARs over 8 days when co-cultured with PSMA-expressing cell lines at E:T of 1:20. b Fold expansion of hPSMA-CARs compared to J591-CAR after 8-day co-culture. c Production of IFNγ at 8 days as determined by ELISA. d Schema of rechallenge with PSMA-CARs co-cultured with PC-3 PSMA lo , PC-3 PSMA hi , or PC-3 PIP and rechallenged with tumor cells every 3 days. e PC-3 PSMA lo (left), PC-3 PSMA hi (center), or PC-3 PIP (right) cell counts were quantified by flow cytometry. b Fold expansion of T cells after each rechallenge were quantified by flow cytometry. c Production of IFNγ by T cells following each rechallenge as determined by ELISA. Data are presented as mean values ± SEM. P values indicate differences between ET260-1-dCH2(28tm)BBz and ET260-1-dCH2(28tm)BBz/mbIL12 and between ET260-1-dCH2(28tm)BBz/mbIL12 and J591-dCH2(4tm)BBz using a two-tailed Student’s t-test.

Article Snippet: Human metastatic prostate cancer cell line PC-3 (ATCC CRL-1435) was cultured in RPMI-1640 (Corning) containing 10% fetal bovine serum (FBS, Hyclone), and 1X antibiotic-antimycotic (AA, Gibco) (complete RPMI).

Techniques: Activity Assay, In Vitro, Cell Culture, Expressing, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Vinculin depletion increases bioenergetics of MDA-MB-231 cells. (A) Representative western blot of MDA-MB-231 cells expressing PercevalHR and pHRed probes transfected with siRNA to knock down vinculin (siVcl), including a scrambled control (siCtrl). (B) Quantification of the PercevalHR ratiometric signal for MDA cells expressing PercevalHR and pHRed probes treated with siRNA to knock down vinculin, including a scrambled control (siCtrl) and siVcl ( N = 3, n = 91–92 cells). (C) Representative ratiometric heatmaps of siCtrl and siVcl MDA cells expressing PercevalHR and pHRed. Scale bar, 100 µm. (D) Mean intensity of 2-NBDG of cells on 2D glass surfaces ( N = 3, n = 70–89 cells). (E) Representative images of 2-NBDG staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (F) Mean intensity of TMRM of cells on 2D glass surfaces ( N = 3, n = 58–84 cells). (G) Representative images of TMRM staining displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 50 µm. (H) Mean intensity of 2-NBDG for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91–103 cells). (I) Representative images of 2-NBDG staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (J) Mean intensity of TMRM for cells embedded in a 1.5 mg/ml collagen matrix ( N = 3, n = 91 cells). (K) Representative images of TMRM staining in a 1.5 mg/ml collagen matrix displayed using a fluorescent channel (top) and the Fire lookup table (bottom) to indicate the relative intensity of the fluorescent signal. Scale bar, 20 µm. (L) Mean intensity of 2-NBDG in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 89–91 cells). (M) Mean intensity of TMRM in NIH/3T3 cells on 2D glass surfaces ( N = 3, n = 90–94 cells). (N) Mean intensity of 2-NBDG in MCF10A cells on 2D glass surfaces ( N = 3, n = 88–90 cells). (O) Mean intensity of TMRM in MCF10A cells on 2D glass surfaces ( N = 3, n = 90–96 cells). (P) Mean intensity of 2-NBDG for MDA ( N = 3, n = 93–98 cells) or Vcl KO ( N = 3, n = 79–86 cells) cells grown either on a tissue culture–treated surface (Adherent) or in a nonadherent plate (Floating), including representative phase-contrast images of MDA or Vcl KO cells seeded on a tissue culture surface or suspended in an ultra-low attachment plate. Scale bar, 50 µm. The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). Bar graphs denote the mean ± SEM. ns = not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. siVcl, siRNA targeting vinculin. Source data are available for this figure: .

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Western Blot, Expressing, Transfection, Knockdown, Control, Staining, Whisker Assay

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Representative western blots. (A) Representative western blot confirming Vcl knockdown in MDA-MB-231 cells expressing PercevalHR and pHRed probes, including a scrambled control (siCtrl) and siVcl. Relevant samples are outlined with a red box, and uncropped membrane is included to show the ladder. (B and C) Representative western blot and (C) quantification from three independent western blots confirming Vcl KO and rescue in MDA-MB-231 cells ( N = 3). The Pre-Sort population refers to the Vcl KO cells transduced with Vcl-Venus before positively expressing Venus cells were sorted using FACS. Bar graph denotes the mean ± SEM. ns = not significant. (D) Representative western blot to confirm Vcl KO in NIH/3T3 cells. (E) Representative western blot to confirm Vcl KO in MCF10A cells. siVcl, siRNA targeting vinculin; FACS, fluorescence-activated cell sorting. Source data are available for this figure: .

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Western Blot, Knockdown, Expressing, Control, Membrane, Transduction, Fluorescence, FACS

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Characterization of the MDA-MB-231 Vcl KO cell line. (A) Quantification of the percentage of dead cells from 30 fields of view per condition using a LIVE/DEAD kit ( N = 3). (B) Representative images of cell lines indicated treated with a LIVE/DEAD kit to assess cell viability in 2D. The right panel represents the positive control condition. Scale bar, 200 µm. (C and D) Percentage of EdU incorporation in parental MDA-MB-231 and Vcl KO cells and (D) representative images of EdU incorporation and DAPI to assess cell proliferation ( N = 3). Scale bar, 100 µm. Graphs denote the mean ± SEM. ns = not significant.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Positive Control

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Vcl KO does not increase migration speed, but reduces persistence of MDA-MB-231 cells. (A) Representative phase-contrast images of cell lines indicated. Scale bar, 100 µm. (B) Quantification of cell aspect ratio and cell spread area on a 2D surface ( N = 3, n > 108 cells). (C and D) Speed and (D) persistence of MDA and Vcl KO cells migrating on 2D surfaces. (E) Representative trajectories of cells moving on 2D surfaces. (F) Representative color-coded time lapses of cells migrating on 2D surfaces where each cell outline represents a 50-min interval from the previous outline. Scale bar, 50 µm. (G) Magnitude of displacement from origin at each time point. Note: some SEM bars are too small to display ( N = 3, n = 78–95 cells for C–G). (H) Quantification of invasive fractions of MDA and Vcl KO cells through collagen-coated transwells ( N = 3, n = 6). (I and J) Speed and (J) persistence of cells migrating in a 1.5 mg/ml collagen matrix ( N = 3, n = 42–52 cells). (K) Representative XY trajectories of cells moving in a 1.5 mg/ml collagen matrix. (L) Cell aspect ratio and spread area in a 1.5 mg/ml collagen matrix ( N = 3, n = 42–52 cells). The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). Bar graphs denote the mean ± SEM. XY plot shows the mean ± SEM. ns = not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Migration, Whisker Assay

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Representative phase-contrast time lapse of an MDA-MB-231 cell migrating in 2D, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse depicts the MDA-MB-231 cell in . Scale bar, 50 µm. Playback, 7 fps.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Inverted Microscopy

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Representative phase-contrast time lapse of an MDA-MB-231 Vcl KO cell migrating in 2D, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse depicts the MDA-MB-231 Vcl KO cell in . Scale bar, 50 µm. Playback, 7 fps.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Inverted Microscopy

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Representative phase-contrast time lapse of an MDA-MB-231 cell moving in a 1.5 mg/ml collagen matrix, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse supports data presented in . Scale bar, 20 µm. Playback, 7 fps.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Inverted Microscopy

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Representative phase-contrast time lapse of an MDA-MB-231 Vcl KO cell moving in a 1.5 mg/ml collagen matrix, acquired on a Zeiss Axio Observer Z1 inverted microscope at 10-min intervals. Time lapse supports data presented in . Scale bar, 20 µm. Playback, 7 fps.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Inverted Microscopy

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Vcl KO increases GTP-RhoA–mediated ROCK activity and cell blebbing in MDA-MB-231 cells. (A) Representative color-coded time lapse of MDA and Vcl KO cell shape on 2D surfaces, where each outline represents a 4-s interval. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (B) Representative time lapse indicating that blebs are forming in the Vcl KO cells as shown by separation of the membrane from the actin cortex labeled using MemGlow and LifeAct, respectively. Scale bar, 10 µm in the first image. Scale bar, 5 µm in the zoomed-in time-lapse images. (C) Representative phase-contrast images of cells treated with 20 µm Y-27632 (Y27) or the vehicle control (deionized water). Scale bar, 100 µm. (D) Representative color-coded time lapse of MDA and Vcl KO cell shape on 2D surfaces treated with 20 µm Y-27632 or the vehicle control. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. For D, each outline represents a 4-s interval. (E) Quantification of the percentage of blebbing cells ( N = 3, n = 28–31 fields of view) and the number of blebs per minute per cell ( N = 3, n = 36 cells) for cells treated with 20 µm Y-27632 or the vehicle control. (F) Quantification of the percentage of blebbing cells in cells treated with 25 µm Blebb or the vehicle control ( N = 3, n = 29 fields of view). (G) Normalized GTP-loaded RhoA measured using absorbance at 490 nm for cells treated with 20 µm Y-27632 or the vehicle control ( N = 4). Bar graphs denote the mean ± SEM. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Activity Assay, Membrane, Labeling, Control

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Representative time lapse of an MDA-MB-231 Vcl KO cell expressing LifeAct (red) and stained with 20 nM MemGlow (green), a membrane dye. The time lapse was acquired on a Zeiss LSM800 confocal microscope at 5-s intervals and represents the MDA-MB-231 Vcl KO cell pictured in . Scale bar, 10 µm. Playback, 3 fps.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Expressing, Staining, Membrane, Microscopy

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Representative time lapse of an MDA-MB-231 cell expressing LifeAct (red) and stained with 20 nM MemGlow (green), a membrane dye. The time lapse was acquired on a Zeiss LSM800 confocal microscope at 5-s intervals and represents the MDA-MB-231 cell pictured in . Scale bar, 10 µm. Playback, 3 fps.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Expressing, Staining, Membrane, Microscopy

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Representative phase-contrast time lapse of MDA-MB-231 cells moving in 2D, treated with vehicle control for Y-27632 (deionized water) for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Control, Inverted Microscopy

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Representative phase-contrast time lapse of MDA-MB-231 Vcl KO cells moving in 2D, treated with vehicle control for Y-27632 (deionized water) for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Control, Inverted Microscopy

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Representative phase-contrast time lapse of MDA-MB-231 cells moving in 2D, treated with 20 µM Y-27632 for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Inverted Microscopy

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Representative phase-contrast time lapse of MDA-MB-231 Vcl KO cells moving in 2D, treated with 20 µM Y-27632 for 20 h. The time lapse was acquired on a Zeiss Axio Observer Z1 inverted microscope at ∼4-s intervals. A still image of this time lapse is provided in . Scale bar, 100 µm. Playback, 7 fps.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Inverted Microscopy

Journal: The Journal of Cell Biology

Article Title: Mechanometabolism of cell adhesion: Vinculin regulates bioenergetics via RhoA-ROCK

doi: 10.1083/jcb.202504025

Figure Lengend Snippet: Metabolic inhibition of Vcl KO cells reduces cell blebbing, and Rho activation of parental MDA-MB-231 cells increases cell blebbing and bioenergetics. (A and B) Representative (A) phase-contrast images (scale bar, 100 µm) and (B) color-coded time lapses of Vcl KO cells treated with oligomycin, iodoacetate, or the vehicle control for each drug. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (C) Percentage of blebbing cells treated with oligomycin ( N = 3, n = 33–34 fields of view), iodoacetate ( N = 3, n = 31 fields of view), or the vehicle and the number of blebs per cell per minute for Vcl KO cells treated with oligomycin ( N = 3, n = 32–33 cells), iodoacetate ( N = 3, n = 32–34 cells), or the vehicle control. (D) Cell spread area of Vcl KO cells treated with oligomycin ( N = 3, n = 92–97 cells), iodoacetate ( N = 3, n = 101–137 cells), or the vehicle control, and the aspect ratio of Vcl KO cells treated with oligomycin ( N = 3, n = 90–91 cells), iodoacetate ( N = 3, n = 94–134 cells), or the vehicle control. (E and F) Representative (E) phase-contrast images (scale bar, 100 µm) and (F) color-coded time lapses of parental MDA cells treated with Rho activator or the vehicle control. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (G) Percentage of blebbing cells ( N = 3, n = 36 fields of view) or number of blebs per cell per minute ( N = 3, n = 36 cells) for MDA cells treated with Rho activator or the vehicle control. (H) Mean intensity of 2-NBDG in parental MDA cells treated with Rho activator or the vehicle control ( N = 3, n = 90–97 cells). (I) Mean intensity of TMRM in parental MDA cells treated with Rho activator or the vehicle control ( N = 3, n = 61–62 cells). (J) Cell spread area ( N = 3, n = 106–140 cells) and aspect ratio ( N = 3, n = 106–140 cells) of parental MDA cells treated with the Rho activator compared with Vcl KO cells. (K) Cell spread area of parental MDA cells being treated with 20 µm oligomycin or the vehicle control over time after trypsinization ( N = 3, n = 74–89 cells). (L) Cell spread area of parental MDA cells being treated with 10 µm iodoacetate or the vehicle control over time after trypsinization ( N = 3, n = 72–87 cells). For K and L, time of 0 h indicates when cells are initially seeded on tissue culture surface and not yet adhered or spread. Bar graphs denote the mean ± SEM. The box-and-whisker plot shows median and 25th/75th percentile (box), min to max (whiskers), and mean (+). XY plots show the mean ± SEM. ns = not significant, **P < 0.01, ****P < 0.0001.

Article Snippet: Highly metastatic MDA-MB-231 breast adenocarcinoma cells (HTB-26; ATCC), HEK-293T cells (CRL-3216; ATCC), and NIH/3T3 fibroblasts (CRL-1658) were maintained at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s media (DMEM; 11965092; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin–streptomycin (Thermo Fisher Scientific).

Techniques: Inhibition, Activation Assay, Control, Whisker Assay

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: Efficiency of lentiviral transduction with il12, il15 and il15Rα or il18 genes and the level of differentiation of bone marrow-derived dendritic cells (DCs) stimulated with B16 F0 tumor antigens (TAg). Concentration of overexpressed cytokines IL-12 (A) , IL-15 (B) , IL-18 (C) in supernatants collected after 48 hours of DCs cultures measured using ELISA. Percentage of CD11c + cells on the 10 th day of DCs cultured (D) . Expression of CD40 (E) , CD80 (F) , CD86 (G) and MHC II (H) molecules on the surface of CD11c + cells (MFI) (bar plots and representative overlay histograms). Results are presented as mean+SD calculated for 5–6 samples per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (A–D, F–H) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (E) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the DC/TAg control cells; crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg control cells, asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ x p<0.05; **/ xx p<0.01; ***/ xxx p<0.001; ****/ xxxx p<0.0001). IC – isotype control , MFI – mean fluorescence intensity .

Article Snippet: The non-metastatic murine melanoma B16 F0 cell line (ECACC 92101204) was cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM, ATCC) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Sigma−Aldrich).

Techniques: Transduction, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Control, Fluorescence

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: The ability of DCs genetically modified to produce IL-12, IL-15/Il-15Rα or IL-18 and stimulated with B16 F0 tumor antigens to prime splenocytes. Percentage of CD8 + cells (A) , CD4 + cells (B) and NK cells (C) among splenocytes obtained after 5-day coculture with DCs. Percentage of effector cells (CD107a + ) among CD8 + (D) , CD4 + (E) and NK cells (F) after 2-hour incubation with B16 F0 cells. Concentration of IFN-γ (G) and IL-10 (H) in supernatants collected after 5 days of cocultured DCs and spleen cells. Ratio of IFN-γ and IL-10 concentration (I) . Cytotoxic activity of splenocytes presented as a percentage of dead B16 F0 tumor cells after 4-hour incubation with effector cells in a ratio of 10:1 (effector:target) (J) . Results are presented as mean+SD calculated for 6–12 samples per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (A–D, G–I) , the parametric one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (E, J) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (F) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the DC/TAg control cells; crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg control cells, asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ x p<0.05; **/ xx p<0.01; ***/ xxx p<0.001; ****/ xxxx p<0.0001).

Article Snippet: The non-metastatic murine melanoma B16 F0 cell line (ECACC 92101204) was cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM, ATCC) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Sigma−Aldrich).

Techniques: Genetically Modified, Incubation, Concentration Assay, Activity Assay, Control

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: Growth of B16 F0 tumors in mice after immunotherapy and chemoimmunotherapy composed of a chemotherapeutic agent, anti-IL-10R antibody, and genetically modified DC-based vaccines stimulated with B16 F0 tumor antigens. Scheme of immunotherapy (A) and chemoimmunotherapy (E) treatment created with BioRender.com . Growth kinetics of B16 F0 tumor in mice treated with immunotherapy (B) or chemoimmunotherapy (F) . Violin plot presenting individual tumor volume and designated median tumor volume for each group, calculated on the 19 th day of the immunotherapy (C) or chemoimmunotherapy (G) experiment. Results are presented as median for 3–10 mice per group. Table presenting B16 F0 tumor growth inhibition (TGI) calculated on the 19 th day of the experiment in relation to the non-treated group (nt) (D, H) . Differences between groups were estimated using the two−way ANOVA followed by Tukey’s multiple comparisons post−hoc test (B, F) or non-parametric Kruskal−Wallis test followed by Dunn’s multiple comparisons test (C, G) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M) – (*p<0.05; **/ ## p<0.01; ***p<0.001; ****p<0.0001).

Article Snippet: The non-metastatic murine melanoma B16 F0 cell line (ECACC 92101204) was cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM, ATCC) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Sigma−Aldrich).

Techniques: Genetically Modified, Vaccines, Inhibition, Control

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: Evaluation of leukocyte subpopulations infiltrating B16 F0 tumor tissue after administration of immunotherapy or chemoimmunotherapy. Scheme of the multiparameter flow cytometry analysis of myeloid and lymphoid cells infiltrating B16 F0 tumor nodules (prepared for one representative sample from group IX in the chemoimmunotherapy experiment) (A) . Percentage of live CD45 + cells in tumor (B, H) . Percentage of each leukocyte’s population among CD45 + cells (C, I) . Percentage of CD8 + (D, J) , CD4 + (E, K) , Treg (F, L) and NK cells (G, M) infiltrating tumor tissue. Results are presented as mean+SD calculated for 3–6 mice per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (B, G, J, M) , the parametric one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (D) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (K, L) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M) ; crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg treated group; asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ # / x p<0.05; **/ xx p<0.01; ***/ xxx p<0.001; ****p<0.0001).

Article Snippet: The non-metastatic murine melanoma B16 F0 cell line (ECACC 92101204) was cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM, ATCC) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Sigma−Aldrich).

Techniques: Flow Cytometry, Control

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: Estimation of TAM and MDSC subpopulation in B16 F0 tumor tissue after applied therapy. Percentage of TAM (A, D) , TAMs MHC II high /TAMs MHC II low (B, E) and MDSC (C, F) among CD45 + cells in tumors. Results are presented as mean+SD calculated for 3–6 mice per group. Differences between groups were estimated using the parametric one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (A, D) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (F) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M); crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg treated group; asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ # p<0.05; **/ ## / xx p<0.01; ***/ xxx p<0.001; ****/ #### p<0.0001).

Article Snippet: The non-metastatic murine melanoma B16 F0 cell line (ECACC 92101204) was cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM, ATCC) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Sigma−Aldrich).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: Impact of applied immunotherapy and chemoimmunotherapy on the induction of systemic antitumor in B16 F0 melanoma model. Scheme of the flow cytometry analysis of restimulated splenocytes (A) . Percentage of CD8 + (B, H) , CD4 + (C, I) , and NK cells (D, J) restimulated splenocytes. Percentage of CD107a + cells among CD8 + (E, K) , CD4 + (F, L) and NK cells (G, M) . Results are presented as mean+SD calculated for 3–6 mice per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (E, H, K) , the parametric one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (B, F, G, I) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (C, D, J, L, M) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M) ; crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg treated group; asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ # p<0.05; **/ ## / xx p<0.01; ***/ ### p<0.001; ****/ #### p<0.0001).

Article Snippet: The non-metastatic murine melanoma B16 F0 cell line (ECACC 92101204) was cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM, ATCC) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Sigma−Aldrich).

Techniques: Flow Cytometry, Control

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: Activity of restimulated splenocytes obtained from B16 F0 melanoma-bearing mice after immunotherapy and chemoimmunotherapy. Concentration of IFN-γ (A, D) , IL-10 (B, E) and IL-4 (C, F) in supernatants after restimulation of spleen cells with B16 F0 cells, measured using ELISA assay. Results are presented as mean+SD calculated for 3–6 mice per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (A, C, F) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (B, D, E) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M); crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg treated group; asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ # p<0.05; **/ ## p<0.01; ***/ ### p<0.001; ****/ #### p<0.0001).

Article Snippet: The non-metastatic murine melanoma B16 F0 cell line (ECACC 92101204) was cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM, ATCC) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Sigma−Aldrich).

Techniques: Activity Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: The effect of chemoimmunotherapy with DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg cell vaccine on the inhibition of B16 F0 tumor growth.

Article Snippet: The non-metastatic murine melanoma B16 F0 cell line (ECACC 92101204) was cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM, ATCC) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Sigma−Aldrich).

Techniques: Inhibition