Journal: Cell Reports Medicine

Article Title: Potentiating immunotherapy in “immune-cold” solid tumors through orchestrating T cell immunity via tumor-specific genetic engineering

doi: 10.1016/j.xcrm.2025.102510

Figure Lengend Snippet: P αCD3&LIGHT mediated co-expression of αCD3 and LIGHT with tumor cell specificity (A) Schematic illustration of P αCD3&LIGHT comprehensively modulating T cell immunity. (B) Representative transmission electron microscopy (TEM) image of DOTAP-PEG-PLGA nanoparticles. Scale bar: 50 nm. (C) Hydrodynamic diameter, polydispersity index (PDI), and zeta potential of DOTAP-PEG-PLGA nanoparticles. n = 3 . (D) Hydrodynamic diameter and polydispersity index (PDI) of DOTAP-PEG-PLGA nanoparticles. n = 3 . (E) Encapsulation efficiency (EE) and drug loading capacity (DLC) of DOTAP-PEG-PLGA nanoparticles for P αCD3&LIGHT . n = 3 . (F) Schematic illustration of the specific expression of αCD3 and LIGHT in tumor cells driven by P αCD3&LIGHT . (G–I) ELISA (G and H) and western blot analysis (I) of the expression of αCD3 and LIGHT in B16-OVA cells transfected with P αCD3&LIGHT or other controls. n = 4 . (J–L) Flow cytometry (J), western blot analysis (K), and representative CLSM images (L) of eGFP expression in lung fibroblasts (MLFs), proximal tubular epithelial cells (PTECs), cytotoxic T lymphocytes (CTLs), Pan B cells, bone marrow-derived dendritic cells (BMDCs), hematopoietic progenitor cells (HPCs), and B16-OVA tumor cells transfected with P αCD3&LIGHT&eGFP or PBS. Nucleus (blue) and eGFP (green). Scale bar: 30 μm. n = 4 . (M) IVIS spectrum images of Cy5 signals in tumors tissues (Tu), heart (H), liver (Li), spleen (S), lung (Lu), kidney (K), lymph node (Ln), brain (Br), stomach (St), intestine (In), and tibia (Ti) of melanoma-bearing mice after intravenous injection of PBS or Cy5-labeled DOTAP-PEG-PLGA nanoparticles. Scale bars: 5 mm. (N) Representative three-dimensional reconstitution images of DOTAP-PEG-PLGA nanoparticles in tumor tissues of melanoma-bearing mice after intravenous injection of Cy5-labeled DOTAP-PEG-PLGA nanoparticles. Nucleus (blue) and DOTAP-PEG-PLGA NPs (red). Scale bar: 15 μm. (O) Bioluminescence images of tumors tissues (Tu), heart (H), liver (Li), spleen (S), lung (Lu), kidney (K), lymph node (Ln), brain (Br), stomach (St), intestine (In), and tibia (Ti) of melanoma-bearing mice after intravenous injection of PBS or P Luc . Scale bars: 5 mm. (P) Representative immunofluorescence images of eGFP-positive cells in tumor tissues of melanoma-bearing mice after intravenous injection of P αCD3&LIGHT&eGFP or PBS. Scale bars: 150 μm. (Q and R) ELISA of αCD3 (Q) and LIGHT (R) in tumors tissues (Tu), heart (H), liver (Li), spleen (S), lung (Lu), kidney (K), lymph node (Ln), brain (Br), stomach (St), intestine (In), and bone marrow (Bm) of melanoma-bearing mice after intravenous injection of P αCD3&LIGHT&eGFP or PBS. n = 4 . Data are represented as mean ± SD (error bars) from biological replicates. p values were determined by one-way ANOVA with Tukey’s test for (G) and (H) and unpaired two-tailed Student’s t test for (J), (Q), and (R). n.s., not significant; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also and .

Article Snippet: Mouse Hydroxyproline (Hyp) ELISA Kit , CUSABIO , CSB-E08839m.

Techniques: Expressing, Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Encapsulation, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Flow Cytometry, Derivative Assay, Injection, Labeling, Immunofluorescence, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Potentiating immunotherapy in “immune-cold” solid tumors through orchestrating T cell immunity via tumor-specific genetic engineering

doi: 10.1016/j.xcrm.2025.102510

Figure Lengend Snippet: P αCD3&LIGHT induced migration of immunocytes into tumor beds (A) Schematic illustration of P αCD3&LIGHT -induced high endothelial venule (HEV) formation and immune cell recruitment. (B) Heatmap of mRNA levels of typical chemokines secreted by cancer-associated fibroblasts (CAFs) following co-incubation with B16-OVA cells transfected with P αCD3&LIGHT or other controls. (C) The numbers of migrated OT-1 cells, Pan B cells, and bone marrow-derived dendritic cells (BMDCs) after co-incubation with CAFs, C166 cells, and B16-OVA cells transfected with P αCD3&LIGHT or other controls. n = 4. (D) ELISA of VEGF-C in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. n = 8. (E) Representative immunofluorescence images of HEVs in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. Nucleus (Blue), CD31-positive vascular endothelial cells (green), PNAd-positive HEV cells (red), and LIGHT (white). Scale bars: 50 μm. (F) Flow cytometry analysis of PNAd-positive HEV cells in CD31-positive vascular endothelial cells after treatment with P αCD3&LIGHT or other controls. n = 8. (G and H) ELISA of MADCAM-1 (G) and GLYCAM-1 (H) in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. n = 8. (I) Heatmap of mRNA levels of chemokines and adhesion molecules in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. (J–N) ELISA of VCAM-1 (J), CCL-5 (K), CCL-19 (L), CXCL-13 (M), and GM-CSF (N) in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. n = 8. (O) Flow cytometry analysis of various immunocytes migrating into tumor microenvironment (TME) after treatment with P αCD3&LIGHT or other controls. Data are represented as mean ± SD (error bars) from biological replicates. p values were determined by one-way ANOVA with Tukey’s test for (C)–(H) and (J)–(N). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also and .

Article Snippet: Mouse Hydroxyproline (Hyp) ELISA Kit , CUSABIO , CSB-E08839m.

Techniques: Migration, Incubation, Transfection, Derivative Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Potentiating immunotherapy in “immune-cold” solid tumors through orchestrating T cell immunity via tumor-specific genetic engineering

doi: 10.1016/j.xcrm.2025.102510

Figure Lengend Snippet: P αCD3&LIGHT facilitated infiltration of immunocytes and formation of tertiary lymphoid structures (A) Schematic illustration of P αCD3&LIGHT -promoted immune cell penetration into tumor parenchyma. (B) Heatmap of mRNA levels of matrix metalloproteinases (MMPs) in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. (C–E) ELISA of MMP-1 (C), MMP-9 (D), and TGF-β (E) in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. n = 8. (F) Quantification of collagen thickness in tumor boundary in the P αCD3&LIGHT or other control groups. n = 20. (G) Representative immunohistochemistry images of Picro-Sirius Red (PS-Red) staining to evaluate collagen fibers in tumor boundary in the P αCD3&LIGHT or other control groups. Scale bar: 200 μm. (H) ELISA of type I collagen in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. n = 8 . (I and J) Representative immunofluorescence images of CD3 + CD8 + T cells in tumor tissues of the melanoma-bearing mice after treatment with PBS (I) or P αCD3&LIGHT (J). Nucleus (blue), CD8 + T cells (green), and CD3 + T cells (red). Scale bars: 200 μm. (K) Schematic illustration of TLS formation induced by LIGHT and its function in fostering stem-like T cells. (L and M) ELISA of CCL-21 (L) and CXCL-12 (M) in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. n = 8. (N and O) Representative immunofluorescence images of TLSs in tumor tissues of the melanoma-bearing mice after treatment with P αCD3&LIGHT or PBS. Nucleus (blue), B220 + B cells (green), and CD3 + T cells (red). Scale bars: 300 μm (original images) or 50 μm (enlarged images). (P–R) The numbers of B cell clusters (P), total TLSs (Q), and deep TLSs (R) in tumor tissues after treatment with P αCD3&LIGHT or other controls. n = 8. (S and T) Flow cytometry analysis of TCF-1-positive stem cell-like CD8 + T cells (S) and Ki67-positive self-renewing CD8 + T cells (T) in the P αCD3&LIGHT or other control groups. n = 8. Data are represented as mean ± SD (error bars) from biological replicates. p values were determined by one-way ANOVA with Tukey’s test for (C)–(F), (H), (L), (M), (S), and (T) and unpaired two-tailed Student’s t test for (P)–(R). n.s., not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Article Snippet: Mouse Hydroxyproline (Hyp) ELISA Kit , CUSABIO , CSB-E08839m.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Immunohistochemistry, Staining, Immunofluorescence, Flow Cytometry, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Potentiating immunotherapy in “immune-cold” solid tumors through orchestrating T cell immunity via tumor-specific genetic engineering

doi: 10.1016/j.xcrm.2025.102510

Figure Lengend Snippet: P αCD3&LIGHT mediated intercellular interaction and T cell activation (A) Schematic illustration of microfluidic model for assessing the interaction between OT-1 cells and B16-OVA cells. (B) Representative three-dimensional reconstruction images and surface renderings illustrating the dynamic trajectories of OT-1 cells at various time intervals. B16-OVA cells (green) and OT-1 cells (red). Scale bar: 30 μm. (C) The percentages of OT-1 cells close to B16-OVA cells transfected with P αCD3&LIGHT or PBS. n = 1,000. (D–F) Track length (D), track straightness (E), and speed (F) of OT-1 cells co-incubated with P αCD3&LIGHT -transfected B16-OVA cells or pristine B16-OVA cells. n = 1,000. (G) Schematic illustration of monitoring intercellular interaction via SrtA-mediated proximity labeling. (H) Representative CLMS images of G 5 peptide on the surface of P αCD3&LIGHT -transfected B16-OVA cells. Nucleus (cyan), αCD3 (green), LIGHT (purple), and G 5 peptide (red). Scale bar: 10 μm. (I) Representative CLMS images of SrtA on the surface of OT-1 cells. Nucleus (blue), SrtA (green), and LPETG peptide (red). Scale bar: 10 μm. (J and K) Representative CLMS and three-dimensional reconstitution images of the interaction between OT-1@SrtA cells and B16-OVA-G 5 cells (J) or B16-OVA cells (K). Nucleus (blue), B16-OVA cells and B16-OVA-G 5 cells (green), and LPETG peptide (red). The white and red arrows indicated the interface of OT-1@SrtA cells with B16-OVA cells or B16-OVA-G 5 cells. Scale bars: 10 μm (CLMS images) or 5 μm (three-dimensional reconstitution images). (L) Flow cytometric analysis of LPETG-positive B16-OVA-G 5 cells transfected with P αCD3&LIGHT or other controls. n = 6. (M) Schematic illustration of T cell activation triggered by the binding of αCD3 to CD3. (N) Heatmap of mRNA levels of activation and exhaustion markers of OT-1 cells following co-incubation with B16-OVA cells transfected with P αCD3&LIGHT or PBS. (O) CCK-8 assay for evaluating the number changes of OT-1 cells in the P αCD3&LIGHT or other control groups. n = 4. (P–R) ELISA of IFN-γ (P), TNF-α (Q), and Gzm-B (R) from cell culture supernatant in the P αCD3&LIGHT or other control groups. n = 4. (S and T) The CCK-8 assay of B16-OVA cell viability (S) and flow cytometry analysis of PI-positive apoptotic B16-OVA cells (T) in the P αCD3&LIGHT or other control groups. n = 4. Data are represented as mean ± SD (error bars) from biological replicates. p values were determined by unpaired two-tailed Student’s t test for (D)–(F) and one-way ANOVA with Tukey’s test for (L) and (O)–(T). ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Article Snippet: Mouse Hydroxyproline (Hyp) ELISA Kit , CUSABIO , CSB-E08839m.

Techniques: Activation Assay, Transfection, Incubation, Labeling, Binding Assay, CCK-8 Assay, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Potentiating immunotherapy in “immune-cold” solid tumors through orchestrating T cell immunity via tumor-specific genetic engineering

doi: 10.1016/j.xcrm.2025.102510

Figure Lengend Snippet: P αCD3&LIGHT mediated suppression of “immune-cold” solid tumors and enhanced therapeutic efficacy of ICIs against melanoma (A and B) Flow cytometry analysis of CD8 + T cells (A) and LPETG-positive B16-OVA-G 5 cells (B) in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. n = 8 . (C and D) Representative immunofluorescence images of LPETG-positive B16-OVA-G 5 cells in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. Nucleus (blue), B16-OVA-G 5 cells (green), and LPETG peptide (red). Scale bars: 50 μm (original images) or 20 μm (enlarged images). (E and F) Flow cytometry analysis of the percentage (E) and count (F) of CD69 + CD8 + activated T cells in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. n = 8 . (G–I) ELISA of IFN-γ (G), TNF-α (H), and Gzm-B (I) in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. n = 8 . (J) Flow cytometric analysis of cleaved caspase-3-positive apoptotic tumor cells in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT or other controls. n = 8 . (K–M) Average tumor growth curves of the melanoma (K)-, colon carcinoma (L)-, and breast cancer (M)-bearing mice treated with P αCD3&LIGHT or other controls. n = 8–10. (N) Flow cytometry analysis of CD69 + CD8 + activated T cells in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT + ICIs or other controls. n = 8 . (O) Therapeutic scheme of P αCD3&LIGHT in combination with immune checkpoint inhibitors (ICIs). Melanoma-bearing mice were treated by four intravenous injections of P αCD3&LIGHT or other controls and four intravenous injections of ICIs or PBS. (P–R) ELISA of IFN-γ (P), TNF-α (Q), and Gzm-B (R) in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT + ICIs or other controls. n = 8 . (S) Flow cytometry analysis of cleaved caspase-3-positive apoptotic tumor cells in tumor tissues of melanoma-bearing mice after treatment with P αCD3&LIGHT + ICIs or other controls. n = 8 . (T–V) Average tumor growth curves (T), tumor weights (U), and survival curves (V) of melanoma-bearing mice after treatment with P αCD3&LIGHT + ICIs or other controls. n = 8–10. Data are represented as mean ± SD (error bars) from biological replicates. p values were determined by one-way ANOVA with Tukey’s test for (A), (B), (E)–(N), and (P)–(U). n.s., not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Article Snippet: Mouse Hydroxyproline (Hyp) ELISA Kit , CUSABIO , CSB-E08839m.

Techniques: Drug discovery, Flow Cytometry, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Potentiating immunotherapy in “immune-cold” solid tumors through orchestrating T cell immunity via tumor-specific genetic engineering

doi: 10.1016/j.xcrm.2025.102510

Figure Lengend Snippet: P αCD3&LIGHT improved anti-melanoma efficacy of CAR-T cells without obvious systemic toxicity (A) Therapeutic scheme of P αCD3&LIGHT in combination with CAR-T cells. hCD19-B16 melanoma-bearing mice were treated with four intravenous injections of P αCD3&LIGHT or other controls and two intravenous injections of CAR-T cells at different doses (0–4 × 10 6 cells per mouse). (B) Experimental timeline of blood serum collection for evaluating severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). (C) Flow cytometry analysis of CAR-T cells in tumor tissues of hCD19-B16 melanoma-bearing mice after treatment with P αCD3&LIGHT + CAR-T cells or other controls. n = 8. (D) Tumor weights of hCD19-B16 melanoma-bearing mice treated with 0–4 × 10 6 CAR-T cells. n = 9 or 10. (E–G) ELISA of SAA (E), IL-6 (F), and IL-1β (G) in blood serum of hCD19-B16 melanoma-bearing mice after treatment with P αCD3&LIGHT + CAR-T cells or other controls. n = 8. (H and I) Body temperature (H) and body weight (I) of hCD19-B16 melanoma-bearing mice after treatment with P αCD3&LIGHT + CAR-T cells or other controls. n = 10. (J) Radar map of fold changes of five CRS-related markers. The larger the area enclosed by the five markers, the more severe the CRS-related symptoms. (K) Representative immunohistochemistry images of CD11b-positive staining to indicate vascular leakage in lung, spleen, and liver tissues from hCD19-B16 melanoma-bearing mice after treatment with P αCD3&LIGHT + CAR-T cells or other controls. Scale bar: 100 μm. (L) Representative immunohistochemistry images of H&E staining to show the thickness of meninges in brain tissues from hCD19-B16 melanoma-bearing mice after treatment with P αCD3&LIGHT + CAR-T cells or other controls. Scale bar: 100 μm. (M) Therapeutic scheme of P αCD3&LIGHT in combination with 1.5 × 10 6 CAR-T cells. hCD19-B16 melanoma-bearing mice were treated with four intravenous injections of P αCD3&LIGHT or other controls and two intravenous injections of PBS or total 1.5 × 10 6 CAR-T cells. (N) Representative IVIS spectrum images of DiR-labeled CAR-T cells at tumor sites in hCD19-B16 melanoma-bearing mice after treatment with P αCD3&LIGHT + 1.5 × 10 6 CAR-T cells or other controls. (O–Q) Flow cytometry analysis of CD69-positive activated CAR-T cells (O), TCF-1-positive stem cell-like (P), and Ki67-positive self-renewing CAR-T cells (Q) in tumor tissues from hCD19-B16 melanoma-bearing mice after treatment with P αCD3&LIGHT + 1.5 × 10 6 CAR-T cells or other controls. n = 8. (R–T) ELISA of IFN-γ (R), TNF-α (S), and Gzm-B (T) in tumor tissues from hCD19-B16 melanoma-bearing mice after treatment with P αCD3&LIGHT + 1.5 × 10 6 CAR-T cells or other controls. n = 8. (U) Flow cytometry analysis of cleaved caspase-3-positive apoptotic hCD19-B16 tumor cells in tumor tissues from hCD19-B16 melanoma-bearing mice after treatment with P αCD3&LIGHT + 1.5 × 10 6 CAR-T cells or other controls. n = 8. (V–X) Individual (V) and average (W) tumor growth curves and survival curves (X) of hCD19-B16 melanoma-bearing mice after treatment with P αCD3&LIGHT + 1.5 × 10 6 CAR-T cells or other controls. n = 8–10. (Y) Magnetic resonance imaging (MRI) images of complete cured melanoma-bearing mice treated with P αCD3&LIGHT + 1.5 × 10 6 CAR-T cells. Data are represented as mean ± SD (error bars) from biological replicates. p values were determined by unpaired two-tailed Student’s t test for (C) and one-way ANOVA with Tukey’s test for (D), (O)–(U), and (W). n.s., not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Article Snippet: Mouse Hydroxyproline (Hyp) ELISA Kit , CUSABIO , CSB-E08839m.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Labeling, Magnetic Resonance Imaging, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Potentiating immunotherapy in “immune-cold” solid tumors through orchestrating T cell immunity via tumor-specific genetic engineering

doi: 10.1016/j.xcrm.2025.102510

Figure Lengend Snippet: LIGHT and αCD3 antibody exhibited clinical therapeutic prospects, and hP αCD3&LIGHT enhanced performance of human CAR-T cells (A–C) The levels of hLIGHT and the numbers of hCD8 + T cells in colorectal cancer (CRC) (A), nasopharyngeal carcinoma (NPC) (B), and cervical cancer (C). n = 12, 10, or 8. (D–F) Regression analysis of hLIGHT expression and the number of hCD8 + T cells in colorectal cancer (CRC) (D), nasopharyngeal carcinoma (NPC) (E), and cervical cancer (F). n = 12, 10, or 8. (G–I) Regression analysis of anti-αCD3 concentration and CD69-positive activated hCAR-T cells (G), proliferative capability (H), and hGzm-B secretion (I). n = 4 . (J and K) ELISA of hαCD3 (J) and hLIGHT (K) in Raji cells transfected with hP αCD3&LIGHT or other controls. n = 4. (L) Representative CLSM images of eGFP expression in Raji cells transfected with hP αCD3&LIGHT&eGFP or PBS. Scale bar: 20 μm. (M and N) ELISA of hαCD3 (M) and hLIGHT (N) in tumor tissues of lymphoma-bearing mice after intravenous injection of hP αCD3&LIGHT&eGFP or PBS. n = 4. (O and P) Heatmaps of the mRNA levels of chemokines and adhesion molecules secreted by hCAFs (O) and HUVECs (P) following co-incubation with Raji cells transfected with hP αCD3&LIGHT or other controls. (Q) Schematic illustration of hP αCD3&LIGHT -mediated recruitment of hCAR-T cells. (R) The number of migrated hCAR-T cells after co-incubating hCAFs, HUVECs, and Raji cells transfected with hP αCD3&LIGHT or other controls. n = 4. (S) Schematic illustration of hP αCD3&LIGHT -mediated activation of hCAR-T cells. (T) Flow cytometric analysis of LPETG-positive Raji-G 5 cells transfected with hP αCD3&LIGHT or other controls after co-incubating with hCAR-T@SrtA cells. n = 6. (U) Flow cytometry analysis of CD69-positive activated hCAR-T cells in the hP αCD3&LIGHT or other control groups. n = 4. (V) CCK-8 assay for evaluating the number changes of hCAR-T cells in the hP αCD3&LIGHT or other control groups. n = 4. (W–Y) ELISA of hIFN-γ (W), hTNF-α (X), and hGzm-B (Y) from cell culture supernatant in the hP αCD3&LIGHT or other control groups. n = 4. (Z) The CCK-8 assay of Raji cell viability in the hP αCD3&LIGHT or other control groups. n = 4. Data are represented as mean ± SD (error bars) from biological replicates. p values were determined by one-way ANOVA with Tukey’s test for (J), (K), (R), and (T)–(Z) and unpaired two-tailed Student’s t test for (M) and (N). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. See also and .

Article Snippet: Mouse Hydroxyproline (Hyp) ELISA Kit , CUSABIO , CSB-E08839m.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Injection, Incubation, Activation Assay, Flow Cytometry, Control, CCK-8 Assay, Cell Culture, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Colony-stimulating factor 3 as a key mediator in the progression of idiopathic pulmonary fibrosis: a novel therapeutic target

doi: 10.1038/s41392-025-02421-6

Figure Lengend Snippet: CSF3 knockout attenuates pulmonary fibrosis progression. a Schematic illustration of the bleomycin (BLM) intratracheal injection (IT) animal model and the procedure used to induce lung fibrosis in C57BL/6 mice. b and c Representative Masson’s trichrome staining ( b , c ) and immunohistochemistry for α-SMA and COL1A1 ( c ) of lung tissue from CSF3 +/+ and CSF3 −/− mice after BLM or PBS treatment ( n = 5 per group). Scale bar: 25 μm, Scale bar: 100 μm. d–i qRT-PCR analysis of α-SMA, COL1A1, and CSF3 expression ( d , e ), hydroxyproline content assay ( f ), qRT-PCR analysis of prolyl hydroxylases (P4HA1, P4HA2, P4HA3) ( g ), matrix metalloproteinase 2 (MMP2) ( h ) and tissue inhibitors of metalloproteinases (TIMP1/2) ( i ) expression in lung tissue from CSF3 +/+ and CSF3 −/− mice after BLM or PBS treatment. j qRT-PCR analysis of α-SMA, COL1A1, and CSF3 expression in human lung fibroblasts isolated from idiopathic pulmonary fibrosis patients (IPDF) and transfected with siRNA targeting CSF3 (si-CSF3). k Western blot analysis of CSF3, αSMA, COL1A1, and p-STAT3 in IPDF cells transfected with si-CSF3 in IPDF. β-Actin was used as a loading control. l Representative immunofluorescence images of α-SMA and COL1A1 in IPDF transfected with si-CSF3. Scale bars: 200 μm. m The spontaneous Matrigel-invading capacity of IPDF transfected with si-CSF3 or si-Control. Scale bars: 200 μm. Statistical significance was determined using ANOVA with multiple comparison or t -test

Article Snippet: Hydroxyproline content in mouse lung tissue or primary lung fibroblasts was quantified using a hydroxyproline assay kit (Chondrex, Inc., #6017) using 10 mg of mouse tissues according to the manufacturer’s instructions.

Techniques: Knock-Out, Injection, Animal Model, Staining, Immunohistochemistry, Quantitative RT-PCR, Expressing, Isolation, Transfection, Western Blot, Control, Immunofluorescence, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Colony-stimulating factor 3 as a key mediator in the progression of idiopathic pulmonary fibrosis: a novel therapeutic target

doi: 10.1038/s41392-025-02421-6

Figure Lengend Snippet: CSF3 induces pulmonary fibrosis through the CSF3R/STAT3 signaling axis. a qRT-PCR analysis of fibrosis markers (α-SMA, COL1A1, fibronectin) in human lung fibroblast cell line (HLF, left) and mouse primary lung fibroblast cells (MLF, right) treated with recombinant CSF3 (rCSF3) (200 ng/ml, 24 h). b and c Representative immunofluorescence images of α-SMA and COL1A1 ( b ), and Western blot analysis of fibrosis markers and STAT3 activation (p-STAT3) ( c ) in HLF and MLF cells treated with rCSF3. β-Actin was used as a loading control. Scale bars: 200 μm. d Hydroxyproline content assay in HLF and MLF cells treated with rCSF3. e Schematic illustration of the procedure used to induce lung fibrosis in C57BL/6 mice with recombinant mouse CSF3. BLM was administered IP on day 1, followed by IP injections of rCSF3 three times per week. f Quantification of the fibrosis area was performed using Orbit software based on Masson’s trichrome staining images. The green area represents the fibrotic region. Scale bar: 500 μm. g Hydroxyproline content in mouse lung tissues from groups treated with BLM and rmCSF3 as indicated. h–k qRT-PCR analysis of α-SMA ( h ), COL1A1 ( i ) and fibronectin ( j ) expression, and hydroxyproline content ( k ) assay in wild-type (CSF3 +/+ ) MLF cells and CSF3 knock-out (CSF3 −/− ) MLF cells treated with BLM and rCSF3. l and m qRT-PCR analysis of fibrosis markers and CSF3 expression ( l ), and representative immunofluorescence images of α-SMA and COL1A1 ( m ) in HLF cells transfected with si-CSF3R and treated with rCSF3 (200 ng/ml, 24 h). Scale bars: 200 μm. n and o Western blot analysis of α-SMA and COL1A1 ( n ), and representative immunofluorescence images of α-SMA and COL1A1 ( o ) in CSF3 −/− MLF cells treated with BLM and rCSF3. β-Actin was used as a loading control. Scale bars: 200 μm. Statistical significance was determined using ANOVA with multiple comparison or t -test

Article Snippet: Hydroxyproline content in mouse lung tissue or primary lung fibroblasts was quantified using a hydroxyproline assay kit (Chondrex, Inc., #6017) using 10 mg of mouse tissues according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Recombinant, Immunofluorescence, Western Blot, Activation Assay, Control, Software, Staining, Expressing, Knock-Out, Transfection, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Colony-stimulating factor 3 as a key mediator in the progression of idiopathic pulmonary fibrosis: a novel therapeutic target

doi: 10.1038/s41392-025-02421-6

Figure Lengend Snippet: Evaluation of CSF3-neutralizing antibody FB-101 in pulmonary fibrosis models. a Schematic representation of the BLM IT injection animal model, the generation of lung fibrosis, and the overall therapeutic procedure in C57BL/6 mice. The mice CSF3 neutralizing antibody (FB-101m) and IgG were administered I.P. b and c Representative H&E ( b ) and Masson’s trichrome staining ( c ) images of lung tissue from each mouse group ( n = 5 per group). Scale bar: 2 mm and 250 μm. d Quantification of the fibrotic area from the Masson’s trichrome staining images using Orbit software. Scale bar: 250 μm. e Picrosirius red-stained therapeutic mouse model samples were analyzed by polarized microscopy. Collagen fibers (left, polarized light) and their subsequent analysis using ImageJ (right, analysis) provide a quantitative assessment of the fibers. Scale bar: 100 μm. f–j qRT-PCR analysis of α-SMA ( f ), COL1A1 ( g ), and CSF3 ( h ) expression, ELISA quantification of CSF3 levels ( i ), and Western blot analysis of CSF3, α-SMA, COL1A1, and fibronectin (FN) ( j ) in lung tissue from each mouse group. β-Actin was used as a loading control. k–n , Hydroxyproline content assay ( k ), qRT-PCR analysis of prolyl hydroxylase family (P4HA1, P4HA2, P4HA3) ( l ), matrix metalloproteinase 2 (MMP2) ( m ), and tissue inhibitors of metalloproteinases (TIMP1/2) ( n ) expression in lung tissue from each mouse group. o–q qRT-PCR analysis of α-SMA, COL1A1, FN, and CSF3 expression ( o ), Western blot analysis of CSF3, α-SMA, COL1A1, and phospho-STAT3 (p-STAT3) ( p ), and representative immunofluorescence images of α-SMA and COL1A1 ( q ) in IPDF treated with the human CSF3 neutralizing antibody (FB-101). β-Actin was used as a loading control. Scale bar: 200 μm. r The spontaneous Matrigel-invading capacity of IPDF treated with FB-101 or IgG. Scale bar: 200 μm. Statistical significance was determined using ANOVA with multiple comparisons or a t -test

Article Snippet: Hydroxyproline content in mouse lung tissue or primary lung fibroblasts was quantified using a hydroxyproline assay kit (Chondrex, Inc., #6017) using 10 mg of mouse tissues according to the manufacturer’s instructions.

Techniques: Injection, Animal Model, Staining, Software, Microscopy, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Immunofluorescence