Journal: bioRxiv

Article Title: Analysis of patient data reveals novel cancer-relevant functions for GCN2/eIF2αK4

doi: 10.1101/2025.03.18.643965

Figure Lengend Snippet: A : GSEA of GCN2-correlating genes. Enriched gene sets (FDR < 0.05) from the MSigDB Hallmark gene set for negatively (left) and positively (right) correlating genes. Selected gene sets indicating possible new functions of GCN2 pursued in this study are shown in bold. The results of the GSEA of the top-ranked 1000 correlating genes in each direction are shown in Table S3. B, C Representative immunoblots to show GCN2 levels in ( B ) HeLa and ( C ) hTert-RPE1 cell lines. Cells were transfected with GCN2-targeting siRNA-s to deplete (siGCN2) or were engineered to stably overexpress GCN2 by lentiviral transduction (GCN2 high ). To estimate the efficiency of depletion, different amounts (100%, 10% and 25%; corresponds to 30, 3 and 7.5 µg protein, respectively) of the lysate from cells transfected with control siRNA was loaded, along with 30 µg each of the transfected and the overexpressing samples. γ-tubulin is shown as a loading control. D, E mRNA levels of selected transcripts in ( D ) HeLa and ( E ) hTert-RPE1 cells. mRNA levels were normalized to TBP and to GCN2 levels in the parental cell line. Each gene was analyzed in at least three independent experiments. The statistical analyses (unpaired t-test, Benjamini, Krieger and Yekutieli method) compared values between the depleted and overexpressing samples, ****p<0.0001, (D) p=0.0879 for CTTN, **p=0.0019 for GSN, *p=0.0111 for NET1, p=0.8585 for MAD2L1, ***p=0.0002 for SMC3, ***p=0.0009 for TUBGCP6, (E) **p=0.0096 for CTTN, **p=0.0025 for FLNA, **p=0.0088 for GSN, p=0.9396 for NET1, *p=0.0128 for MAD2L1, *p=0.0149 for SMC3, **p=0.0037 for NEK2, **p=0.0020 for TUBGCP6.

Article Snippet: Antibodies rabbit pericentrin 1:400 (Abcam, #ab4448); sheep α/β-tubulin 1:500 (Cytoskeleton Inc ATN02)

Techniques: Western Blot, Transfection, Stable Transfection, Transduction, Control

Journal: bioRxiv

Article Title: Analysis of patient data reveals novel cancer-relevant functions for GCN2/eIF2αK4

doi: 10.1101/2025.03.18.643965

Figure Lengend Snippet: A, B Progression through mitosis in the presence of GCN2i at the indicated concentrations was observed by live-cell imaging in A SiHa and B Caski cells. The time in mitosis is shown for each cell. Prophase was judged by the first frame showing chromatin condensation), anaphase was scored based on chromosome separation and mitotic slippage was scored based on decondensation of the DNA in the absence of chromosome separation. Brown Forsythe and Welch Anova test was performed on the total time in mitosis. SiHa 0 µM compared to 1 µM p=0.1236; 2 µM *p=0.0014; 3 µM ****p<0.0001. Caski 0 µM compared to 1 µM p=0.0551; 2µM and 3 µM **** p<0.0001. C Caski and SiHa cells were incubated in the absence (control) or presence (GCN2i) of GCN2i for 8 hours and fixed for immunofluorescence. Representative images of mitotic cells stained for tubulin (magenta), pericentrin (PCNT, cyan) and DNA (grey) are shown. Scale bars represent 5 µm. D High levels of GCN2 give no protection in starved cells. Hela cells transduced to overexpress GCN2 (GCN2 high ) and the parental cell line (Ctr) were grown in the presence of HFG at the indicated concentrations and observed in Incucyte. Data shown are from three independent experiments. Mean ± SEM are shown, non-linear regression, p=0.9867 for 0 nM, p=0.1607 for 25 nM, p=0.9012 for 50 nM, p=0.1994 for 75 nM, p=0.3283 for 100 nM. E High levels of GCN2 gives protection in cells exposed to MPS1i. Hela cells transduced to overexpress GCN2 (GCN2 WT ) and the parental cell line (Ctr) were grown in the presence of MPS1i at the indicated concentrations and observed in Incucyte. Data shown are from four independent experiments. Mean ± SEM are shown, non-linear regression, p=0.3393 for 0 µM, ****p<0.0001 for 3 µM, 4 µM, and 5 µM. F Protection in cells exposed to MPS1i is lost in cells expressing GCN2 RAXA . HeLa cells transduced to express siRNA-resistant GCN2 RAXA were transfected with GCN2-targeting siRNA (GCN2 RAXA ), grown in the presence of MPS1i at the indicated concentrations, and observed in Incucyte. Data shown are from four independent experiments. Mean and SEM are shown, non-linear regression, p= 0.5069 for 0 µM, p= 0.1346 for 3 µM, p=0.4104 for 4 µM, p=0.4254 for 5 µM. Note that the data shown for control cells are the same as those shown in .

Article Snippet: Antibodies rabbit pericentrin 1:400 (Abcam, #ab4448); sheep α/β-tubulin 1:500 (Cytoskeleton Inc ATN02)

Techniques: Live Cell Imaging, Incubation, Control, Immunofluorescence, Staining, Expressing, Transfection

Journal: bioRxiv

Article Title: Analysis of patient data reveals novel cancer-relevant functions for GCN2/eIF2αK4

doi: 10.1101/2025.03.18.643965

Figure Lengend Snippet: A Migration capacity correlates with GCN2 levels in hTert-RPE1 cells. hTert-RPE1 cells were transfected with GCN2-targeting siRNA for 48 h (siGCN2) or transduced to stably overexpress GCN2 (GCN2 high ). Cells were seeded without FBS into transwell chambers with 8 µM pore size and exposed to an FBS gradient for 16 hours. Migrated cells were counted after DAPI staining and normalized to seeding controls. Three independent experiments, mean and STDEV are shown. B Immunoblots of lysates of cells from the experiment shown in A. γ-tubulin is used as a loading control. C hTert-RPE1 cells were transfected with GCN2-targeting siRNA (siGCN2) or treated with 2 µM GCN2i (GCN2i). Cells were grown to confluence and wounds were created using a Wound Maker tool (Sartorius) and healing was monitored in Incucyte. Three (siGCN2) or five (GCN2i) independent experiments, mean ± SEM are shown, non-linear regression, ****p<0.0001 D HeLa cells treated with 2 µM GCN2i (GCN2i) or transduced to stably overexpress GCN2 (GCN2 high ) were seeded into Ibidi culture inserts and grown to confluence. After removal of the inserts the cells were observed by live-cell imaging. The number of cells migrating into the initial wound area is shown. Three (GCN2i) or four (GCN2 High ) independent experiments, mean and SEM are shown, non-linear regression, ****p<0.0001 E Representative images from an experiment shown in (D).

Article Snippet: Antibodies rabbit pericentrin 1:400 (Abcam, #ab4448); sheep α/β-tubulin 1:500 (Cytoskeleton Inc ATN02)

Techniques: Migration, Transfection, Stable Transfection, Pore Size, Staining, Western Blot, Control, Live Cell Imaging

Journal: bioRxiv

Article Title: Analysis of patient data reveals novel cancer-relevant functions for GCN2/eIF2αK4

doi: 10.1101/2025.03.18.643965

Figure Lengend Snippet: A , hTert-RPE1 and B, HeLa cells were grown to confluence and wounds were created using a Wound Maker tool (Sartorius) and healing was monitored in Incucyte. ISRIB or GCN2i was added to 200 nM or 2 µM, respectively. Four (hTert-RPE) or three (HeLa) independent experiments, mean and SEM are shown, non-linear regression, ****p<0.0001 C hTert-RPE1 cells transduced with GCN2-siR carrying the indicated mutations were transfected with control or GCN2-targeting siRNA for 48 h. Cells were seeded without FBS into transwell chambers with 8 µM pore size and exposed to an FBS gradient for 16 hours. Migrated cells were counted after DAPI staining and normalized to seeding controls, then to migration in cells transfected with control siRNA. Mean ± SEM are shown, results from at least three independent experiments. One-way Anova,****p<0.0001 siCtr versus siGCN2; **p=0.0022 siCtr versus GCN2 Wt siCtr; **p=0.0051 GCN2 Wt siGCN2 versus GCN2 RAXA siGCN2; **p=0.0025 GCN2 Wt siGCN2 versus GCN2 K619R siGCN2 D Knock-down efficiency and expression of the mutant transgenes in the experiments shown in C was tested by immunoblotting. γ-tubulin is shown as loading control. E hTert-RPE1 cells transduced with doxycycline-inducible siRNA-resistant GCN2 carrying the indicated mutations were transfected with control (siCtr) or GCN2-targeting siRNA (siGCN2) and grown to confluence in the presence of doxycycline. Wounds were created using a Wound Maker tool (Sartorius) and healing was monitored in Incucyte. ISRIB was added to 200 nM after wounding. Averages (lines) and SEM (bands) from six independent experiments are shown. Non-linear regression, **p=0.0028, ***p=0.0002, **** p<0.0001. F HeLa cells transduced with doxycycline-inducible siRNA-resistant GCN2 carrying the indicated mutations were transfected with control (siCtr) or GCN2-targeting siRNA (siGCN2), seeded into Ibidi culture inserts, and grown to confluence in the presence of doxycycline. After removal of the inserts the cells were observed by live-cell imaging. ISRIB was added to 200 nM after removal of the inserts. The number of cells migrating into the initial wound area is shown. Averages (lines) and SEM (bands) from three independent experiments are shown. Non-linear regression, ****p<0.0001, *p=0.0113, **p=0.0072, ***p=0.0006 G, H Metastatic potential (G) and penetrance (H) in metastatic cancers as a function of GCN2 mRNA levels. Data were downloaded from depmap.org and are based on a study by Jin et al (2020), which reported metastatic-potential profiling of ca. 500 human cancer cell lines derived from 21 types of solid tumour in immunodeficient murine models . Metastatic potential is calculated based on the mean cancer-cell numbers detected in the target organs. Metastatic penetrance refers to the proportion of mice displaying metastases with the given cell line .

Article Snippet: Antibodies rabbit pericentrin 1:400 (Abcam, #ab4448); sheep α/β-tubulin 1:500 (Cytoskeleton Inc ATN02)

Techniques: Transduction, Transfection, Control, Pore Size, Staining, Migration, Knockdown, Expressing, Mutagenesis, Western Blot, Live Cell Imaging, Derivative Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: A Novel Hydrogen Sulfide Donor Reduces Pilocarpine-Induced Status Epilepticus and Regulates Microglial Inflammatory Profile

doi: 10.3389/fncel.2021.780447

Figure Lengend Snippet: The H 2 S donor downregulated the levels of microglial pro-inflammatory profile in vivo . (A,B) Representative images of iNOS (red) with Iba1 (green) immunostaining or (C,D) Arg1 (red) with Iba1 (green) immunostaining in the CA1 and CA3 regions of the hippocampus in control and the H 2 S donor-treated SE mice. Scale bar 25 μm in the merged panel and 5 μm in the magnified panel. Magnified images are expansions of boxed areas in corresponding panels in the left of each magnified image. (E) Quantitative analysis of Iba1 + cells in CA1 and CA3 regions of the hippocampus. (F) Quantitative data are shown the ratio of iNOS + : Iba1 + to total Iba1 + cells. n = 6 per group. *** p < 0.001 vs SE. Two-way ANOVA with Bonferroni post hoc tests. (G) Quantitative data are shown as the ratio of Arg1 + : Iba1 + to total Iba1 + cells. n = 6 per group. *** p < 0.001 vs SE. Student’s t test. (H–K) Western blot assay shows that expression levels of microglial pro-inflammatory markers (COX2 and TNF-α) are increased in the hippocampus of SE mice. However, the H 2 S donor decreased COX2 and TNF-α expression and increased Arg-1and IL-10 expression in SE mice. The results are expressed as the mean±SEM. n = 3 per group.* p < 0.05, ** p < 0.01 vs Control, # p < 0.05, ## p < 0.01 vs SE. One-way ANOVA with Bonferroni post hoc tests.

Article Snippet: Then, the membranes were blocked with bovine serum albumin (BSA), and incubated with rabbit anti-COX2 (1:500, #12375-1-AP, Proteintech Group, United States), rabbit anti-Arg-1 (1:4,000, #16001-1-AP, Proteintech Group, United States), rabbit anti-TNF-α (1:1,000, #bs-0078R, BIOSS, China), rabbit anti-IL-10 (1:1,000, #bs-20373R, BIOSS, China), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:8,000, #60004-1-Ig, Proteintech Group, United States), and rabbit anti-Tubulin (1:1,000, #11224-1-AP, Proteintech Group, United States) at 4°C overnight.

Techniques: In Vivo, Immunostaining, Western Blot, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: A Novel Hydrogen Sulfide Donor Reduces Pilocarpine-Induced Status Epilepticus and Regulates Microglial Inflammatory Profile

doi: 10.3389/fncel.2021.780447

Figure Lengend Snippet: The H 2 S donor downregulated the levels of microglial pro-inflammatory profile in vitro . (A–C,E) Immunofluorescent staining and quantification of iNOS and Arg1 in lipopolysaccharide (LPS)-treated BV2 cells. Scale bars, 50 μm for the original images and 10 μm for the magnified images. n = 6 per group. *** p < 0.001 vs Control, # p < 0.05, ### p <0.001 vs LPS. Statistical significance was determined by two-way ANOVA with Bonferroni post hoc tests. (D,F) Expression levels of microglial pro-inflammatory markers (COX2, TNF-α) and microglial anti-inflammatory markers (Arg1, IL-10) in BV2 cells were determined by Western blotting. The H 2 S donor decreased the expression of pro-inflammatory markers and upregulated the expression of anti-inflammatory markers in LPS-induced inflammation model of microglia. n = 3 per group. * p < 0.05, ** p < 0.01 vs Control, # p < 0.05, ## p < 0.01 vs LPS. One-way ANOVA with Bonferroni post hoc tests.

Article Snippet: Then, the membranes were blocked with bovine serum albumin (BSA), and incubated with rabbit anti-COX2 (1:500, #12375-1-AP, Proteintech Group, United States), rabbit anti-Arg-1 (1:4,000, #16001-1-AP, Proteintech Group, United States), rabbit anti-TNF-α (1:1,000, #bs-0078R, BIOSS, China), rabbit anti-IL-10 (1:1,000, #bs-20373R, BIOSS, China), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:8,000, #60004-1-Ig, Proteintech Group, United States), and rabbit anti-Tubulin (1:1,000, #11224-1-AP, Proteintech Group, United States) at 4°C overnight.

Techniques: In Vitro, Staining, Expressing, Western Blot

Journal: EBioMedicine

Article Title: DsbA-L interacts with VDAC1 in mitochondrion-mediated tubular cell apoptosis and contributes to the progression of acute kidney disease

doi: 10.1016/j.ebiom.2022.103859

Figure Lengend Snippet: Attenuation of IR-induced renal injury, tubular cell apoptosis, and mitochondrion damage, and the expression of Bax, Cyt-c, and cleaved caspase3 in PT-DsbA-L-KO mice. The bilateral renal arteries of PT-DsbA-L-KO and PT-DsbA-L-WT littermate mice were clamped for 28min and then released for 48 h to establish an IR model. (a) BUN (b) Serum creatinine (c) Hematoxylin and eosin staining. (d) Representative sections of TUNEL-positive cells. (e) Electron microscope detection of mitochondrion morphology. (f) Tubular damage score. (g) The number of TUNEL-positive cells. (h) Mitochondria injury score. (i) The immunoblot analysis of the expression of cleaved caspase3 and DsbA-L, and the expression of BAX and Cyt-c in mitochondrial and cytosolic fractions. β-tubulin and GAPDH was used as whole lysate or cytosolic loading control, respectively. COX IV was used as mitochondria loading control. (j–o) Analysis of the gray scale image between them. (p and q) RT-qPCR detection of the expression of NRF1 and PCG-1α. (r) The immunoblot analysis of the expression of NRF1 and PCG-1α. (s and t) Analysis of the gray scale image between them. Original magnification x 400 or x6000. Scar Bar:100µM in c&d 20µM in e. Each experiment(c–e, i and r) was repeated 6 times independently with similar results. One-way ANOVA was used for the Multiple group comparisons. (a, b,f–h, j–o, p–q & s–t). # p<0.05 : versus saline group, sham group, or MCD group. # P< 0.05 versus sham group. * P< 0.05 versus PT-DsbA-L-WT with IR group.

Article Snippet: Anti-Bax (50599-2-Ig, RRID: AB_2061561) , Cyt-c (10993-1-AP, RRID:AB_2090467) , NRF1 (12482-1-AP, RRID: AB_2282876), GAPDH (60004-1-Ig, RRID: AB_2107436), and β-tubulin (10094-1-AP, RRID: AB_2210695) antibodies were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Expressing, Staining, TUNEL Assay, Microscopy, Western Blot, Control, Quantitative RT-PCR, Saline

Journal: EBioMedicine

Article Title: DsbA-L interacts with VDAC1 in mitochondrion-mediated tubular cell apoptosis and contributes to the progression of acute kidney disease

doi: 10.1016/j.ebiom.2022.103859

Figure Lengend Snippet: DsbA-L-mediated the IR-induced expression of cleaved caspase3, BAX, and Cyt-c in BUMPT cells . The plasmid and siRNA of DsbA-L were transfected into BUMPT cells and then exposed to ATP depletion for 2 h and recovery for 2 h. (a&n) Flow cytometry analysis of BUMPT cells apoptosis. (b&o) The immunoblots analysis the expression of cleaved caspase3 and DsbA-L, and the expression of BAX and Cyt-c in mitochondrial and cytosolic fractions. β-tubulin and GAPDH was used as whole lysate or cytosolic loading control, respectively. COX IV was used as mitochondria loading control. (c–h and q–u) Analysis of the gray scale image between them. (i,j and v, w) RT-qPCR detection the expression of NRF1 and PCG-1α. (k and x) The immunoblot analysis of the expression of NRF1 and PCG-1α. (l, m and y,z) Analysis of the gray scale image between them. Each experiment(a, b, k, n, o and x) was repeated 6 times independently with similar results. One-way ANOVA was used for the Multiple group comparisons. (c–j,c–m, p–w & y, z). # p<0.05 : versus saline group, sham group, or MCD group. # P<0.05 versus scramble with saline group. * P<0.05 versus scramble with IR group.

Article Snippet: Anti-Bax (50599-2-Ig, RRID: AB_2061561) , Cyt-c (10993-1-AP, RRID:AB_2090467) , NRF1 (12482-1-AP, RRID: AB_2282876), GAPDH (60004-1-Ig, RRID: AB_2107436), and β-tubulin (10094-1-AP, RRID: AB_2210695) antibodies were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Expressing, Plasmid Preparation, Transfection, Flow Cytometry, Western Blot, Control, Quantitative RT-PCR, Saline

Journal: BioMed Research International

Article Title: Explore the Effect of Asthma Regulating HIF-1 Pathway on Sperm Quality Based on Rat Model

doi: 10.1155/2022/4194685

Figure Lengend Snippet: (a) Analyses of testicular tissue in mice using HE staining. (b) Electrophoretogram of three proteins (IL6, Stat3, p-Stat3, and HIF-1 α ) in rats from the C, M groups. (c) The expression levels of three proteins (IL6, Stat3, p-Stat3, and HIF-1 α ) in rats from the C, M groups determined using western blotting. (d) The mRNA expression levels of three proteins (IL6, Stat3, and HIF-1 α ) in rats from the C, M groups determined using RT-qPCR. Values are the mean ± SEM ( n = 6 animals per group). The t -test was used. Group M was compared with group C, ∗ represents P < 0.05, ∗∗ represents P < 0.01.

Article Snippet: Drugs and reagents used included the following: Ovalbumin (Sigma, A5503; MO, USA); Aluminium hydroxide gel (Pierce, 77161; SH, CN); BCA protein quantitative kit (Chinese British Institute of Biotechnology, SI-5700; BJ, CN); c-DNA Kit (Thermo, #1622; MA, USA); IL6 antibody (Bioss, bs-6309R; Woburn, MA, USA); p-Stat3 antibody (Bioss, bs-1658R; Woburn, MA, USA); HIF-1 α antibody (Bioss, bs-20398R; Woburn, MA, USA); Compression atomizer (Omron, NE-C900; DL, CN); and Stainless steel cell sieve (Puyi Biotechnology, PY-0643; SH, CN).

Techniques: Staining, Expressing, Western Blot, Quantitative RT-PCR

Journal: eLife

Article Title: A survey of optimal strategy for signature-based drug repositioning and an application to liver cancer

doi: 10.7554/eLife.71880

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Collagen I (Rabbit polyclonal) , ProteinTech , Cat#: 14695-1-AP; RRID: AB_2082037 , WB (1:2000)IF (1:200).

Techniques: Sequencing, Software

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: Mice bearing Cer2-OVA MMTV-PyMT lung metastases were treated with vehicle (0.5% HPMC) or 75 mg/kg VS-4718 p.o. for 2 weeks starting 4 weeks post-implantation. ( A & B ) Representative images and quantification of collagen VIIIα1 and laminin 5α in metastatic versus non-metastatic lung regions at 6 weeks (H-score; each dot represents one lesion; n = 5 mice/group). Kruskal-Wallis test and Dunn’s multiple comparison. ( C & D ) Collagen VIIIα1 and laminin 511 suppress CD8⁺ T cell activation in vitro . CD8⁺ T cells were plated on 1 μg/mL ECM components, stimulated with CD3/CD28 + IL-2 for 48 h, and assessed by flow cytometry for CD25, granzyme B, IFNγ, and TNFα expression (n = 3-5). ( E ) ECM-mediated inhibition of CD8⁺ T cell migration. Transwell inserts coated with laminin 511, or collagen VIIIα were used to assess migration toward serum-rich media for 5 h (n = 3-4). ( F ) ECM-dependent CD8⁺ T cell adhesion. Adhesion to laminin 511, or collagen VIIIα-coated plates was quantified after 90 min and normalized to uncoated plates (n = 3-4). (G & H) Kaplan-Meier curves showing progression-free survival (PFS) of patients with invasive breast carcinoma stratified by (G) COL8A1 and (H) LAMA5 expression levels. Patients were divided into high and low expression groups based on the median RNA-seq expression. Point-wise z-test using Greenwood’s standard error. Data source = Breast invasive carcinoma TCGA. ( I-K ) Spearman’s Correlation of Metastatic burden signature correlated to FAK-dependent ECM score (I ), COL8A1 ( J ) and LAMA5 ( K ) expression in triple negative breast cancer patients of the SCAN-B dataset . Spearman’s correlation of cytotoxic CD8 + T cells with COL8A1 ( L ) and LAMA5 ( M ) expression in triple negative breast cancer patients of the SCAN-B dataset. ( L & M ). Log 2 mRNA abundance of COL8A1 and LAMA5 in triple negative breast cancer patients with complete response (pCR) or residual disease (RD) in biopsies taken before treatment with either Palclitaxel and Pembrolizumab ( O ) or Palclitaxel, ABT888 and Carboplatin ( N ) in ISPY-2 trial.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Comparison, Activation Assay, In Vitro, Flow Cytometry, Expressing, Inhibition, Migration, RNA Sequencing

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: Mice carrying Cer2-OVA MMTV-PyMT metastatic lesions or without Cer2-OVA MMTV-PyMT injection were treated for two weeks with either vehicle control (0.5% HPMC) or VS-4718 (75 mg/kg). Quantification (H-score) of concentration of (A) Collagen VIIIα1 and (B) Laminin 5α in metastatic lesions and tumor adjacent areas at 5 weeks post implantation of Cer2-OVA MMTV-PyMT cells. Each dot represents a metastatic lesion. n = 5 mice/group using whole lung slide scans. One-way ANOVA for normal distributed with Tukey’s multiple comparison test. Mean ± SEM; ** = p < 0.01.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Injection, Control, Concentration Assay, Comparison

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: (A) Representative images of collagen VIIIα1 in metastatic lesions. Scalebar = 50 μm. (B) Quantification (H-score) of collagen VIIIα1 in metastatic lesions and tumor adjacent areas. (C) Representative images of laminin 5 α in metastatic lesions and tumor adjacent areas. Scalebar = 50 μm. (D) Quantification (H-score) of laminin 5 α in metastatic lesions and tumor adjacent areas. Dots represent metastatic lesions. n = 5 mice/group using whole lung slide scans. Kruskal-Wallis test and Dunn’s multiple comparison for not normal distributed. Mean ± SEM; **** = p < 0.0001.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Comparison

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: (A) Mean migration velocity and (B) directional change rate/ average turning frequency of activated CD8⁺ T cells migrating on laminin 511 or collagen VIIIα1 (1 µg/cm²). T cells were CellTracker™ Deep Red-labeled and imaged for 6 h at 5-min intervals; tracks with >11 spots were analyzed using TrackMate . Laminin-511 reduced velocity and increased turning frequency, whereas collagen VIIIα1 had no significant effect. n = 3 independent experiments with 3-5 field of views per group. Unpaired t-tests. Mean ± SEM; **** = p < 0.0001.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Migration, Labeling

Journal: bioRxiv

Article Title: FAK Inhibition Remodels the Metastatic ECM and Restores CD8⁺ T Cell Trafficking and Immunosurveillance

doi: 10.64898/2026.01.21.700837

Figure Lengend Snippet: FAK inhibition reduces laminin-α5 and collagen VIIIα1 within metastatic lesions, weakening basement-membrane-derived physical and inhibitory barriers, thereby improving CD8⁺ T-cell infiltration, migration, tumor-cell engagement, and cytotoxic activity to promote metastatic regression.

Article Snippet: Slides were incubated overnight at 4 °C with primary antibodies against laminin α5 (polyclonal, Bioss, Cat# BS-1086R, 1:500) or collagen VIIIα1 (rabbit polyclonal, Abcam, Cat# ab236653, 1:300) diluted in antibody diluent.

Techniques: Inhibition, Membrane, Derivative Assay, Migration, Activity Assay