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ATCC murine embryonic fibroblasts
Murine Embryonic Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 912 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 912 article reviews

murine embryonic fibroblasts - by Bioz Stars, 2026-06

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Journal: Cell reports

Article Title: Radiation-induced autophagy regulates fibroblast mitochondrial metabolism and crosstalk with triple-negative breast cancer cells

doi: 10.1016/j.celrep.2026.117096

Figure Lengend Snippet: (A) Representative transmission electron microscopy (TEM) images of unirradiated (0 Gy) and irradiated (10 Gy) 3T3 fibroblasts 7 days after radiation therapy (RT). N, nucleus; ER, endoplasmic reticulum; M, mitochondria; and LD, lipid droplet. Arrows indicate mitochondria. Scale bars, 800 nm. (B) Representative immunofluorescence (IF) images of ATP5A1 (green) and nuclear (blue) staining at 7 days after RT. Scale bars, 10 μm. (C) Example binary images of mitochondria used for aspect ratio analysis. Images were selected to show how small changes in aspect ratio result in large changes in elongation. Scale bars, 5 μm. (D) Quantification of the mitochondrial aspect ratio at 3 and 7 days after RT in unirradiated and irradiated 3T3 fibroblasts. Each data point represents mitochondria from individual cells with n = 4 independent replicates. (E) Quantification of the total area of ATP5A1 staining per cell in 3- and 7-day post-RT fibroblasts ( n = 4). (F) Mitochondrial DNA (mtDNA) copy number analysis of 3- and 7-day post-RT 3T3 fibroblasts ( n = 3). (G) Representative images of human immortalized mammary fibroblasts (iMFs) with ATP5A1 (green) and nuclear (blue) staining 7 days after RT. Scale bars, 10 μm. (H) Quantification of the mitochondrial aspect ratio at 7 days after RT in unirradiated and irradiated iMFs. Each data point represents mitochondria from individual cells with n = 3 independent replicates. (I) Relative gene expression of Mfn2 in irradiated and unirradiated murine 3T3 fibroblasts incubated with an siRNA for Mfn2 (siMfn2) or a control sequence (siCtrl) 7 days after RT ( n = 3). (J) Representative IF images of ATP5A1 (green) and nuclear (blue) staining 7 days after RT in irradiated and unirradiated 3T3 fibroblasts after Mfn2 knockdown using siRNA. Scale bars, 10 μm. (K) Quantification of the mitochondrial aspect ratio 7 days after RT in unirradiated and irradiated 3T3 fibroblasts with or without siRNA knockdown of Mfn2 . Each data point represents mitochondria from individual cells with n = 3 independent replicates. Statistical analysis was performed as follows: for (D)–(F), (I), and (K), a two-way ANOVA with Tukey’s multiple comparisons test for differences of means with * p < 0.05, ** p < 0.01, and *** p < 0.001 and for (H), an unpaired two-tailed t test with *** p < 0.001. Error bars show standard deviation. See also and .

Article Snippet: NIH 3T3 murine embryonic fibroblasts were obtained from ATCC (CRL-1658), and immortalized human reduction mammoplasty fibroblasts (iMFs) were obtained from Dr. Charlotte Kuperwasser (Tufts University) in April 2022.

Techniques: Transmission Assay, Electron Microscopy, Irradiation, Immunofluorescence, Staining, Gene Expression, Incubation, Control, Sequencing, Knockdown, Two Tailed Test, Standard Deviation

(A) Representative TEM images of unirradiated and irradiated 3T3 fibroblasts 7 days after RT, highlighting the differences in vacuole-like structures. V, vacuole-like structure; N, nucleus; M, mitochondria; and LD, lipid droplet. Scale bars, 800 nm. (B) Representative images of LAMP-1 (yellow) and nuclear (blue) staining of unirradiated and irradiated 3T3 fibroblasts 7 days after RT. Scale bars, 30 μm. (C) Quantification of LAMP-1 area per cell in irradiated and unirradiated 3T3 fibroblasts 7 days after RT ( n = 6). (D) Representative western blot for ATG5 with GAPDH as loading control for both irradiated and unirradiated fibroblasts at 3 and 7 days after RT. (E) Quantification of relative protein expression of ATG5 normalized to GAPDH loading control expression in irradiated and unirradiated fibroblasts ( n = 3). (F) Representative images of LC3B (green) and nuclear (blue) staining in irradiated and unirradiated 3T3s 3 days after RT following autophagy inhibition with chloroquine (CQ). Autophagosomes are identified by the formation of bright LC3B puncta. Scale bars, 10 μm. (G) Corresponding quantification of relative autophagosome area per cell, normalized to the biological replicate average, in irradiated and control 3T3s at 3 and 7 days after RT ( n = 6, 3-day; n = 5, 7-day). (H) Representative western blot showing p62 expression with α-tubulin loading control for 3T3s at both 3 and 7 days after RT with and without CQ incubation. (I) Quantification of p62 protein expression relative to α-tubulin loading control in irradiated and unirradiated fibroblasts ( n = 3). Statistical analysis was performed as follows: for (C), an unpaired two-tailed t test with * p < 0.05 and for (E), (G), and (I), a two-way ANOVA with Tukey’s multiple comparisons test for differences of means with * p < 0.05, ** p < 0.01, and *** p < 0.001. Error bars show standard deviation. See also .

Journal: Cell reports

Article Title: Radiation-induced autophagy regulates fibroblast mitochondrial metabolism and crosstalk with triple-negative breast cancer cells

doi: 10.1016/j.celrep.2026.117096

Figure Lengend Snippet: (A) Representative TEM images of unirradiated and irradiated 3T3 fibroblasts 7 days after RT, highlighting the differences in vacuole-like structures. V, vacuole-like structure; N, nucleus; M, mitochondria; and LD, lipid droplet. Scale bars, 800 nm. (B) Representative images of LAMP-1 (yellow) and nuclear (blue) staining of unirradiated and irradiated 3T3 fibroblasts 7 days after RT. Scale bars, 30 μm. (C) Quantification of LAMP-1 area per cell in irradiated and unirradiated 3T3 fibroblasts 7 days after RT ( n = 6). (D) Representative western blot for ATG5 with GAPDH as loading control for both irradiated and unirradiated fibroblasts at 3 and 7 days after RT. (E) Quantification of relative protein expression of ATG5 normalized to GAPDH loading control expression in irradiated and unirradiated fibroblasts ( n = 3). (F) Representative images of LC3B (green) and nuclear (blue) staining in irradiated and unirradiated 3T3s 3 days after RT following autophagy inhibition with chloroquine (CQ). Autophagosomes are identified by the formation of bright LC3B puncta. Scale bars, 10 μm. (G) Corresponding quantification of relative autophagosome area per cell, normalized to the biological replicate average, in irradiated and control 3T3s at 3 and 7 days after RT ( n = 6, 3-day; n = 5, 7-day). (H) Representative western blot showing p62 expression with α-tubulin loading control for 3T3s at both 3 and 7 days after RT with and without CQ incubation. (I) Quantification of p62 protein expression relative to α-tubulin loading control in irradiated and unirradiated fibroblasts ( n = 3). Statistical analysis was performed as follows: for (C), an unpaired two-tailed t test with * p < 0.05 and for (E), (G), and (I), a two-way ANOVA with Tukey’s multiple comparisons test for differences of means with * p < 0.05, ** p < 0.01, and *** p < 0.001. Error bars show standard deviation. See also .

Techniques: Irradiation, Staining, Western Blot, Control, Expressing, Inhibition, Incubation, Two Tailed Test, Standard Deviation

(A) Representative TEM images of unirradiated and irradiated 3T3 fibroblasts treated with CQ 7 days after RT. V, vacuole-like structure; M, mitochondria; LD, lipid droplet; and ER, endoplasmic reticulum. Black arrows indicate higher magnification of mitochondria. Scale bars, 1 μm. (B) Representative IF images of ATP5A1 (green) and nuclear (blue) staining 7 days after RT with and without CQ treatment in irradiated and unirradiated 3T3 fibroblasts. Scale bars, 10 μm. (C) Quantification of the mitochondrial aspect ratio at 3 and 7 days after RT in unirradiated and irradiated 3T3 fibroblasts with and without CQ. Each data point represents mitochondria from one cell ( n = 3). (D) Representative western blot of ATG5 protein expression compared to GAPDH loading control in irradiated and unirradiated shScr and shAtg5 3T3 fibroblasts. (E and F) Representative western blot of LC3B-I and LC3B-II protein expression compared to α-tubulin loading control in irradiated and unirradiated shScr and shAtg5 fibroblasts 7 days after RT with and without CQ incubation to determine changes in autophagic flux, with corresponding quantification of relative LCB-II to α-tubulin expression in (F) ( n = 3). (G) Relative gene expression of Mfn2 in shScr and shAtg5 fibroblasts 3 and 7 days after RT (3-day n = 3, 7-day n = 5). (H) Representative IF images of ATP5A1 (green) and nuclear (blue) staining at 7 days after RT in irradiated and unirradiated shScr and shAtg5 fibroblasts. Scale bars, 10 μm. (I) Quantification of the mitochondrial aspect ratio at 3 and 7 days after RT in unirradiated and irradiated shScr and shAtg5 fibroblasts. Each data point represents mitochondria from one cell ( n = 3). For (C), (F), (G), and (I), statistical significance was determined using a two-way ANOVA with Tukey’s multiple comparisons test for differences of means with * p < 0.05, ** p < 0.01, and *** p < 0.001. Error bars show standard deviation. See also .

Journal: Cell reports

Article Title: Radiation-induced autophagy regulates fibroblast mitochondrial metabolism and crosstalk with triple-negative breast cancer cells

doi: 10.1016/j.celrep.2026.117096

Figure Lengend Snippet: (A) Representative TEM images of unirradiated and irradiated 3T3 fibroblasts treated with CQ 7 days after RT. V, vacuole-like structure; M, mitochondria; LD, lipid droplet; and ER, endoplasmic reticulum. Black arrows indicate higher magnification of mitochondria. Scale bars, 1 μm. (B) Representative IF images of ATP5A1 (green) and nuclear (blue) staining 7 days after RT with and without CQ treatment in irradiated and unirradiated 3T3 fibroblasts. Scale bars, 10 μm. (C) Quantification of the mitochondrial aspect ratio at 3 and 7 days after RT in unirradiated and irradiated 3T3 fibroblasts with and without CQ. Each data point represents mitochondria from one cell ( n = 3). (D) Representative western blot of ATG5 protein expression compared to GAPDH loading control in irradiated and unirradiated shScr and shAtg5 3T3 fibroblasts. (E and F) Representative western blot of LC3B-I and LC3B-II protein expression compared to α-tubulin loading control in irradiated and unirradiated shScr and shAtg5 fibroblasts 7 days after RT with and without CQ incubation to determine changes in autophagic flux, with corresponding quantification of relative LCB-II to α-tubulin expression in (F) ( n = 3). (G) Relative gene expression of Mfn2 in shScr and shAtg5 fibroblasts 3 and 7 days after RT (3-day n = 3, 7-day n = 5). (H) Representative IF images of ATP5A1 (green) and nuclear (blue) staining at 7 days after RT in irradiated and unirradiated shScr and shAtg5 fibroblasts. Scale bars, 10 μm. (I) Quantification of the mitochondrial aspect ratio at 3 and 7 days after RT in unirradiated and irradiated shScr and shAtg5 fibroblasts. Each data point represents mitochondria from one cell ( n = 3). For (C), (F), (G), and (I), statistical significance was determined using a two-way ANOVA with Tukey’s multiple comparisons test for differences of means with * p < 0.05, ** p < 0.01, and *** p < 0.001. Error bars show standard deviation. See also .

Techniques: Irradiation, Staining, Western Blot, Expressing, Control, Incubation, Gene Expression, Standard Deviation

$(A and B) Representative flow cytometry analysis of irradiated and unirradiated 3T3 fibroblasts stained with MitoTracker Deep Red (A) with and without CQ incubation 7 days after RT, with (B) corresponding mean intensity quantification ( n = 4). (C and D) Representative flow cytometry analysis of irradiated and unirradiated 3T3 fibroblasts stained with MitoTracker Deep Red (C) in shScr and shAtg5 fibroblasts 7 days after RT with (D) corresponding mean intensity quantification ( n = 3). (E and F) ATP-linked oxygen consumption rate (OCR) in irradiated and unirradiated 3T3s (E) with and without CQ incubation ( n = 3) and (F) in shScr and shAtg5 fibroblasts 7 days after RT ( n = 3). (G) ATP-linked OCR in irradiated and unirradiated 3T3s 7 days after RT with and without etomoxir incubation to evaluate fatty acid oxidation (FAO) ( n = 3). (H) Schematic showing tricarboxylic acid (TCA) cycle intermediate labeling from a fully labeled 13 C-palmitate tracing study. The first turn of the TCA cycle is depicted. (I) Quantification of the M+2 fractions of the TCA cycle intermediates citrate, succinate, and malate 6 h following 13 C-labeled palmitate introduction at the 7-day post-RT time point ( n = 3). (J) Baseline extracellular acidification rate (ECAR) in irradiated and unirradiated 3T3s 7 days after RT ( n = 3). (K) Glycolytic ECAR in irradiated and unirradiated 3T3s 7 days after RT ( n = 3). (L) Relative L-lactate secretion rate in irradiated and unirradiated 7-day post-RT 3T3 fibroblasts ( n = 6). (M) Quantification of L-lactate concentration in fibroblast CM collected 7 days after RT from irradiated and unirradiated 3T3s ( n = 5). Statistical analysis was performed as follows: for (B) and (D)–(G), a two-way ANOVA with Tukey’s multiple comparisons test for differences of means with * p < 0.05, ** p < 0.01, and *** p < 0.001; for (I), (J), (L), and (M), an unpaired two-tailed t test with * p < 0.05 and ** p < 0.01; and for (K), a paired two-tailed t test with * p < 0.05. Error bars show standard deviation. See also , , and .$

Journal: Cell reports

Article Title: Radiation-induced autophagy regulates fibroblast mitochondrial metabolism and crosstalk with triple-negative breast cancer cells

doi: 10.1016/j.celrep.2026.117096

Figure Lengend Snippet: (A and B) Representative flow cytometry analysis of irradiated and unirradiated 3T3 fibroblasts stained with MitoTracker Deep Red (A) with and without CQ incubation 7 days after RT, with (B) corresponding mean intensity quantification ( n = 4). (C and D) Representative flow cytometry analysis of irradiated and unirradiated 3T3 fibroblasts stained with MitoTracker Deep Red (C) in shScr and shAtg5 fibroblasts 7 days after RT with (D) corresponding mean intensity quantification ( n = 3). (E and F) ATP-linked oxygen consumption rate (OCR) in irradiated and unirradiated 3T3s (E) with and without CQ incubation ( n = 3) and (F) in shScr and shAtg5 fibroblasts 7 days after RT ( n = 3). (G) ATP-linked OCR in irradiated and unirradiated 3T3s 7 days after RT with and without etomoxir incubation to evaluate fatty acid oxidation (FAO) ( n = 3). (H) Schematic showing tricarboxylic acid (TCA) cycle intermediate labeling from a fully labeled 13 C-palmitate tracing study. The first turn of the TCA cycle is depicted. (I) Quantification of the M+2 fractions of the TCA cycle intermediates citrate, succinate, and malate 6 h following 13 C-labeled palmitate introduction at the 7-day post-RT time point ( n = 3). (J) Baseline extracellular acidification rate (ECAR) in irradiated and unirradiated 3T3s 7 days after RT ( n = 3). (K) Glycolytic ECAR in irradiated and unirradiated 3T3s 7 days after RT ( n = 3). (L) Relative L-lactate secretion rate in irradiated and unirradiated 7-day post-RT 3T3 fibroblasts ( n = 6). (M) Quantification of L-lactate concentration in fibroblast CM collected 7 days after RT from irradiated and unirradiated 3T3s ( n = 5). Statistical analysis was performed as follows: for (B) and (D)–(G), a two-way ANOVA with Tukey’s multiple comparisons test for differences of means with * p < 0.05, ** p < 0.01, and *** p < 0.001; for (I), (J), (L), and (M), an unpaired two-tailed t test with * p < 0.05 and ** p < 0.01; and for (K), a paired two-tailed t test with * p < 0.05. Error bars show standard deviation. See also , , and .

Techniques: Flow Cytometry, Irradiation, Staining, Incubation, Labeling, Concentration Assay, Two Tailed Test, Standard Deviation

(A) Non-mitochondrial OCR in irradiated and unirradiated 3T3s at 3 and 7 days after RT ( n = 3). (B and C) Representative flow cytometry histogram analysis of MitoSOX staining in unirradiated and irradiated 3T3 fibroblasts 3 and 7 days after RT with and without CQ treatment. The corresponding mean MitoSOX intensity quantification is shown in (C). (D and E) Representative flow cytometry histogram analysis of MitoSOX staining in unirradiated and irradiated shScr and shAtg5 cells 7 days after RT. The corresponding mean MitoSOX intensity quantification is shown in (E). (F) Multiplex Luminex analysis of upregulated cytokines and chemokines in CM from irradiated and unirradiated 3T3 fibroblasts 7 days after RT ( n = 3). (G) Relative gene expression of Il6 at 3 and 7 days after RT in shScr and shAtg5 fibroblasts ( n = 3). (H) Kaplan-Meier analysis of overall survival utilizing normal adjacent tissue (NAT) data from the TCGA-BRCA dataset accessed through UCSC’s Xena platform , and grouping patients based on high or low gene expression using upper and lower quartiles of MAP1LC3C and IL6 . Statistical analysis was performed as follows: for (A), (C), (E), and (G), a two-way ANOVA with Tukey’s multiple comparisons test for differences of means with * p < 0.05, ** p < 0.01, and *** p < 0.001; for (F), an unpaired two-tailed t test for irradiated versus unirradiated cytokine pairs with * p < 0.05, ** p < 0.01, and *** p < 0.001; and for (H), a log rank test. Error bars show standard deviation. See also , and .

Journal: Cell reports

Article Title: Radiation-induced autophagy regulates fibroblast mitochondrial metabolism and crosstalk with triple-negative breast cancer cells

doi: 10.1016/j.celrep.2026.117096

Figure Lengend Snippet: (A) Non-mitochondrial OCR in irradiated and unirradiated 3T3s at 3 and 7 days after RT ( n = 3). (B and C) Representative flow cytometry histogram analysis of MitoSOX staining in unirradiated and irradiated 3T3 fibroblasts 3 and 7 days after RT with and without CQ treatment. The corresponding mean MitoSOX intensity quantification is shown in (C). (D and E) Representative flow cytometry histogram analysis of MitoSOX staining in unirradiated and irradiated shScr and shAtg5 cells 7 days after RT. The corresponding mean MitoSOX intensity quantification is shown in (E). (F) Multiplex Luminex analysis of upregulated cytokines and chemokines in CM from irradiated and unirradiated 3T3 fibroblasts 7 days after RT ( n = 3). (G) Relative gene expression of Il6 at 3 and 7 days after RT in shScr and shAtg5 fibroblasts ( n = 3). (H) Kaplan-Meier analysis of overall survival utilizing normal adjacent tissue (NAT) data from the TCGA-BRCA dataset accessed through UCSC’s Xena platform , and grouping patients based on high or low gene expression using upper and lower quartiles of MAP1LC3C and IL6 . Statistical analysis was performed as follows: for (A), (C), (E), and (G), a two-way ANOVA with Tukey’s multiple comparisons test for differences of means with * p < 0.05, ** p < 0.01, and *** p < 0.001; for (F), an unpaired two-tailed t test for irradiated versus unirradiated cytokine pairs with * p < 0.05, ** p < 0.01, and *** p < 0.001; and for (H), a log rank test. Error bars show standard deviation. See also , and .

Techniques: Irradiation, Flow Cytometry, Staining, Multiplex Assay, Luminex, Gene Expression, Two Tailed Test, Standard Deviation

(A) Schematic representing the time course of conditioned media (CM) collection for a 4T1 TNBC cell scratch assay. (B and C) Representative images of 4T1 TNBC cells in scratch assays at the 0 and 15 h time points following incubation in either irradiated or unirradiated control fibroblast CM. The corresponding quantification ( n = 6) is shown in (C). (D) Schematic representing the time course of the scratch assay with 4T1 TNBC cells co-cultured with either irradiated or unirradiated 3T3 fibroblasts. (E and F) Representative images of 4T1 TNBC cells (red) in scratch assays co-cultured with irradiated or unirradiated 3T3 fibroblasts (unlabeled) at the 0 and 25 h time points. The corresponding quantification (n = 3–4) is shown in (F). (G) Schematic representing the time course of CM collection for the 3D 4T1 TNBC cell tumorsphere assay. (H and I) Representative images of 4T1 TNBC tumorspheres after 9 days of incubation in either irradiated or unirradiated control fibroblast CM. The corresponding quantification ( n = 30 tumorspheres) is shown in (I). (J and K) Representative images of 4T1 TNBC tumorspheres after 9 days of incubation in either irradiated or unirradiated shScr or shAtg5 fibroblast CM. The corresponding quantification ( n = 18 tumorspheres) is shown in (K). (L and M) Representative images of 4T1 TNBC tumorspheres after 9 days of incubation in either irradiated or unirradiated fibroblast CM with or without an IL-6 neutralizing antibody. The corresponding quantification ( n = 25–26 tumorspheres) is shown in (M). (N) Schematic representing the time course of shScr and shAtg5 fibroblast irradiation and co-culture with 4T1 TNBC cells in the tumorsphere assay. (O and P) Representative images of 4T1 TNBC tumorspheres after 10 days of co-culture with irradiated or unirradiated shScr or shAtg5 fibroblasts. The corresponding quantification ( n = 16 tumorspheres) is shown in (P). Statistical analysis was performed as follows: for (C) and (F), an unpaired two-tailed t test with * p < 0.05 and ** p < 0.01; for (I), a Mann-Whitney test for non-normally distributed distributions with ** p < 0.01; and for (K), (M), and (P), a two-way ANOVA with Tukey’s multiple comparisons test for differences of means with * p < 0.05, ** p < 0.01, and *** p < 0.001. Scale bars are 200 μm in (B) and (E) and 500 μm in (H), (J), (L), and (M). Error bars show standard deviation.

Journal: Cell reports

Article Title: Radiation-induced autophagy regulates fibroblast mitochondrial metabolism and crosstalk with triple-negative breast cancer cells

doi: 10.1016/j.celrep.2026.117096

Figure Lengend Snippet: (A) Schematic representing the time course of conditioned media (CM) collection for a 4T1 TNBC cell scratch assay. (B and C) Representative images of 4T1 TNBC cells in scratch assays at the 0 and 15 h time points following incubation in either irradiated or unirradiated control fibroblast CM. The corresponding quantification ( n = 6) is shown in (C). (D) Schematic representing the time course of the scratch assay with 4T1 TNBC cells co-cultured with either irradiated or unirradiated 3T3 fibroblasts. (E and F) Representative images of 4T1 TNBC cells (red) in scratch assays co-cultured with irradiated or unirradiated 3T3 fibroblasts (unlabeled) at the 0 and 25 h time points. The corresponding quantification (n = 3–4) is shown in (F). (G) Schematic representing the time course of CM collection for the 3D 4T1 TNBC cell tumorsphere assay. (H and I) Representative images of 4T1 TNBC tumorspheres after 9 days of incubation in either irradiated or unirradiated control fibroblast CM. The corresponding quantification ( n = 30 tumorspheres) is shown in (I). (J and K) Representative images of 4T1 TNBC tumorspheres after 9 days of incubation in either irradiated or unirradiated shScr or shAtg5 fibroblast CM. The corresponding quantification ( n = 18 tumorspheres) is shown in (K). (L and M) Representative images of 4T1 TNBC tumorspheres after 9 days of incubation in either irradiated or unirradiated fibroblast CM with or without an IL-6 neutralizing antibody. The corresponding quantification ( n = 25–26 tumorspheres) is shown in (M). (N) Schematic representing the time course of shScr and shAtg5 fibroblast irradiation and co-culture with 4T1 TNBC cells in the tumorsphere assay. (O and P) Representative images of 4T1 TNBC tumorspheres after 10 days of co-culture with irradiated or unirradiated shScr or shAtg5 fibroblasts. The corresponding quantification ( n = 16 tumorspheres) is shown in (P). Statistical analysis was performed as follows: for (C) and (F), an unpaired two-tailed t test with * p < 0.05 and ** p < 0.01; for (I), a Mann-Whitney test for non-normally distributed distributions with ** p < 0.01; and for (K), (M), and (P), a two-way ANOVA with Tukey’s multiple comparisons test for differences of means with * p < 0.05, ** p < 0.01, and *** p < 0.001. Scale bars are 200 μm in (B) and (E) and 500 μm in (H), (J), (L), and (M). Error bars show standard deviation.

Techniques: Wound Healing Assay, Incubation, Irradiation, Control, Cell Culture, Co-Culture Assay, Two Tailed Test, MANN-WHITNEY, Standard Deviation

GC cells increase collagen 1 expression in fibroblasts and collagen increases proliferation of GC cells (A) Expressions of Col1A1, Col1A2, and ACTA2 mRNA in mouse gastric cancer (GC) cells (T3-2D), human GC cells (MKN45, MKN7, and NUGC4), mouse fibroblast cells (MEF), and human fibroblast cells (YS-1 and FEF3) are shown. All cells were analyzed by quantitative RT-PCR analysis. Data are expressed as the mean ± SD ( n = 3). (B) Expression of Col1A1 and Col1A2 mRNA in MEF (upper) and YS-1 (lower) cells after incubation with serum-free medium (SFM) as a control and conditioned medium (CM) of each GC cell type for 4 days. Cells were analyzed using quantitative RT-PCR analysis. Data are expressed as the mean ± SD ( n = 3). (C) Representative images of immunocytochemical staining of collagen 1 and α-SMA in MEF (left) and YS-1 (right) after incubation with normal medium and CM of each of the GC cells for 4 days. SFM was used as a control. The area index for each staining was evaluated by ImageJ software. Data are expressed as the mean ± SD ( n = 3). Scale bars, 50 μm. (D) Cell proliferation of T3-2D and MKN45 cells with and without 10% collagen stimulation for 72 h. Data are expressed as the mean ± SD ( n = 5). (E) Representative cellular morphological images of T3-2D and MKN45 cells with and without 10% collagen 1 stimulation for 72 h. Scale bar, 100 μm. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Journal: Molecular Therapy Oncology

Article Title: Collagen depletion by pirfenidone enhances antitumor effect of oncolytic adenovirus against peritoneal metastases of gastric cancer

doi: 10.1016/j.omton.2025.201045

Figure Lengend Snippet: GC cells increase collagen 1 expression in fibroblasts and collagen increases proliferation of GC cells (A) Expressions of Col1A1, Col1A2, and ACTA2 mRNA in mouse gastric cancer (GC) cells (T3-2D), human GC cells (MKN45, MKN7, and NUGC4), mouse fibroblast cells (MEF), and human fibroblast cells (YS-1 and FEF3) are shown. All cells were analyzed by quantitative RT-PCR analysis. Data are expressed as the mean ± SD ( n = 3). (B) Expression of Col1A1 and Col1A2 mRNA in MEF (upper) and YS-1 (lower) cells after incubation with serum-free medium (SFM) as a control and conditioned medium (CM) of each GC cell type for 4 days. Cells were analyzed using quantitative RT-PCR analysis. Data are expressed as the mean ± SD ( n = 3). (C) Representative images of immunocytochemical staining of collagen 1 and α-SMA in MEF (left) and YS-1 (right) after incubation with normal medium and CM of each of the GC cells for 4 days. SFM was used as a control. The area index for each staining was evaluated by ImageJ software. Data are expressed as the mean ± SD ( n = 3). Scale bars, 50 μm. (D) Cell proliferation of T3-2D and MKN45 cells with and without 10% collagen stimulation for 72 h. Data are expressed as the mean ± SD ( n = 5). (E) Representative cellular morphological images of T3-2D and MKN45 cells with and without 10% collagen 1 stimulation for 72 h. Scale bar, 100 μm. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: A murine embryonic fibroblast (MEF) cell line was purchased from the American Type Culture Collection (Manassas, VA) and maintained in DMEM supplemented with 15% FBS.

Techniques: Expressing, Quantitative RT-PCR, Incubation, Control, Staining, Software

Collagens and fibroblasts inhibit oncolytic virus penetration and reduce antitumor effects (A) T3-2D, MKN45, MEF, and YS-1 cells were infected with OBP-702 at the indicated MOIs for 3 days. Cell viability was assessed using the XTT assays. Cell viability was calculated relative to that of the mock-infected cells, which were set as 1.0. Data are expressed as the mean ± SD ( n = 5). (B) T3-2D and MKN45 cells were infected with OBP-702 at the indicated MOIs for 3 days with or without 10% collagen containing medium. Cell viability was assessed using the XTT assays. Cell viability was calculated relative to that of the mock-infected cells, which were set as 1.0. Data are expressed as the mean ± SD ( n = 5). (C) Representative cellular morphological images of T3-2D and MKN45 cells infected with 100 MOI of OBP-702 with or without 10% collagen stimulation for 72 h. Scale bars, 100 μm. (D) Representative spheroid morphological images of T3-2D cells infected with 20 MOI of OBP-702 or MKN45 cells infected with 50 MOI of OBP-702 with or without 2% collagen stimulation for 7 days. Scale bars, 100 μm. (E) ATP cell viability assay of T3-2D and MKN45 spheroids with or without 2% collagen stimulation for 7 days after infection with OBP-702 (20 MOI or 50 MOI). Data are expressed as the mean ± SD ( n = 5). (F) Representative images of mono-spheroids and co-cultured spheroids infected with OBP-401 (100 MOI) for 48 h. T3-2D and MKN45 cells were labeled with red cell trackers and MEF and YS-1 cells with blue cell trackers. Scale bars, 50 μm. ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Journal: Molecular Therapy Oncology

Article Title: Collagen depletion by pirfenidone enhances antitumor effect of oncolytic adenovirus against peritoneal metastases of gastric cancer

doi: 10.1016/j.omton.2025.201045

Figure Lengend Snippet: Collagens and fibroblasts inhibit oncolytic virus penetration and reduce antitumor effects (A) T3-2D, MKN45, MEF, and YS-1 cells were infected with OBP-702 at the indicated MOIs for 3 days. Cell viability was assessed using the XTT assays. Cell viability was calculated relative to that of the mock-infected cells, which were set as 1.0. Data are expressed as the mean ± SD ( n = 5). (B) T3-2D and MKN45 cells were infected with OBP-702 at the indicated MOIs for 3 days with or without 10% collagen containing medium. Cell viability was assessed using the XTT assays. Cell viability was calculated relative to that of the mock-infected cells, which were set as 1.0. Data are expressed as the mean ± SD ( n = 5). (C) Representative cellular morphological images of T3-2D and MKN45 cells infected with 100 MOI of OBP-702 with or without 10% collagen stimulation for 72 h. Scale bars, 100 μm. (D) Representative spheroid morphological images of T3-2D cells infected with 20 MOI of OBP-702 or MKN45 cells infected with 50 MOI of OBP-702 with or without 2% collagen stimulation for 7 days. Scale bars, 100 μm. (E) ATP cell viability assay of T3-2D and MKN45 spheroids with or without 2% collagen stimulation for 7 days after infection with OBP-702 (20 MOI or 50 MOI). Data are expressed as the mean ± SD ( n = 5). (F) Representative images of mono-spheroids and co-cultured spheroids infected with OBP-401 (100 MOI) for 48 h. T3-2D and MKN45 cells were labeled with red cell trackers and MEF and YS-1 cells with blue cell trackers. Scale bars, 50 μm. ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: A murine embryonic fibroblast (MEF) cell line was purchased from the American Type Culture Collection (Manassas, VA) and maintained in DMEM supplemented with 15% FBS.

Techniques: Virus, Infection, Viability Assay, Cell Culture, Labeling

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