Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Identification of FZD4, FZD6, and TGFBR1 as miR-101 targets. a, 3′-UTR reporter assay: FZD4-UTR-S1 (position 1–1,432) containing miR-101-binding site 1 (position 537–543), FZD4-UTR-S2 (position 4,178–5,463) containing miR-101-binding site 2 (position 1,240–1,246), full length of FZD6-UTR, TGFBR1-UTR-S1 (position 66–1,524) containing miR-101-binding site 1 (position 460–466), or TGFBR1-UTR-S2 (position 3,210–4,749) containing miR-101-binding site 2 (position 3,993–3,999) were co-transfected into HEK 293T cells with miR-101-1. The relative luciferase activities were measured using the Dual-Luciferase® reporter assay system. n = 3. b and c, effect of overexpressing or knockdown miR-101 on the protein expression of FZD4, FZD6, and TGFBR1 in fibroblasts. LL29 cells were treated with a lentivirus expressing miR-101-1 or the virus control (VC) at an m.o.i. of 50 for 48 h (b) or CCD-8Lu fibroblasts were treated with a lentivirus expressing anti-miR-101 or the virus control (Anti-CON) at an m.o.i. of 50 for 72 h. c, Western blotting was performed to determine the protein expression of FZD4, FZD6, and TGFBR1. The protein samples used here were the same as mentioned in Fig. 7c. The GAPDH immunoblot shown in Fig. 7c was used again here. d, overexpression of miR-101 inhibits FZD4, FZD6, and TGFBR1 mRNA expression in fibroblasts. LL29 cells were treated with lentiviral miR-101 or the virus control at an m.o.i. of 50 for 48 h. The mRNA expression of FZD4, FZD6, and TGFBR1 was determined by real-time PCR and normalized to GAPDH. The results are presented as the mean ± S.E. n = 3. e, real-time PCR showing the increased mRNA levels of FZD4, FZD6, and TGFBRR1 by anti-miR-101. CCD-8Lu fibroblasts were treated with lentiviral anti-miR-101 or the virus control at an m.o.i. of 50 for 72 h. The expression levels were relative to GAPDH from two cell preparations, each performed in duplicate. f–h, mRNA expression of FZD4, FZD6, and TGFBR1 in IPF patient lungs. The mRNA levels of FZD4, FZD6, and TGFBR1 were determined by real-time PCR and normalized to β-actin. n = 8–10. The results are presented as the mean ± S.E. Statistical analyses were performed by using Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Reporter Assay, Binding Assay, Transfection, Luciferase, Knockdown, Expressing, Virus, Control, Western Blot, Over Expression, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Overexpression of FZD4, FZD6, or TGFBR1 rescues miR-101-mediated inhibition of fibroblast proliferation and activation. a–d, knockdown of TGFBR1 repressed the TGF-β1-induced mRNA expression of α-SMA, COL1A1, COL3A1, and COL4A1 in fibroblasts. LL29 cells were treated with lentivirus shRNA-TGFBR1 or the virus control at an m.o.i. of 50 for 48 h. Then, the cells were stimulated with 5 ng/ml TGF-β1 for 48 h. The α-SMA, COL1A1, COL3A1, and COL4A1 mRNA expression levels were determined by real-time PCR. The expression levels were relative to GAPDH from two cell preparations, each performed in duplicate. The results are presented as the mean ± S.D. **, p < 0.01. e and f, overexpression of FZD4 or FZD6 rescues the miR-101-mediated inhibition of fibroblast proliferation. LL29 cells were split into 96-well plates at 3,000 cells per well. After a 24-h culture, cells were infected with lentiviruses at a total of m.o.i. = 50 (miR-Con miR-101, GFP, FZD4, and FZD6 at m.o.i. = 25 each, virus control (VC) = miR-Con + GFP) for 48 h. Cells were starved for another 24 h and stimulated with WNT5a (1 μg/ml) for 12 h. FZD4 and FZD6 expression was determined by Western blotting by using anti-GFP antibody. Cell proliferation was determined by BrdU assay. Data were presented as mean ± S.E. Statistical analysis was performed by ANOVA and followed by Tukey's HSD test. n = 4. **, p < 0.01. g–j, overexpression of TGFBR1 rescues miR-101-mediated inhibition of α-SMA, COL1A1, and COL3A1 mRNA expression. LL29 cells were infected with lentiviruses at a total of m.o.i. = 50 (miR-Con, miR-101, GFP, and TGFBR1 at m.o.i. = 25 each, virus control = miR-Con + GFP) for 48 h. Cells were then stimulated with TGFβ1 (5 ng/ml) for 48 h. Cells were collected for real-time PCR. The results are presented as the mean ± S.E., n = 3. ANOVA followed by Tukey's HSD test was performed. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Over Expression, Inhibition, Activation Assay, Knockdown, Expressing, shRNA, Virus, Control, Real-time Polymerase Chain Reaction, Infection, Western Blot, BrdU Staining

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Adenovirus-mediated miR-101 gene transfer attenuates bleomycin-induced pulmonary fibrosis. On day 1, an adenovirus expressing miR-101-1 or the control adenovirus (VC) (5 × 109 pfu per mouse) was delivered into the lungs of 6–8-week-old C57BL/6 mice by nasal instillation. On day 2, bleomycin (Bleo) (0.06 units per mouse) or the same volume of saline (Sal) was intranasally instilled into the lungs. On day 15, the mice were subjected to the analysis of respiratory mechanics by Flexivent and then sacrificed. The left lungs were collected for RNA and protein analysis, and the right lungs were fixed for histological analysis. a, real-time PCR analysis showing adenovirus-mediated overexpression of miR-101 in the mouse lung. The expression levels were relative to U6. b, H&E staining showing the fibrotic changes in the mouse lung induced by bleomycin. miR-101 attenuated the fibrotic changes in bleomycin-treated mouse lungs. Scale bar, 100 μm. c, Ashcroft score grade of pulmonary fibrosis. d, lung collagen content as determined using the QuickZyme hydroxyproline assay kit. e and f, real-time PCR analysis showing the increased COL1A1 and COL3A1 mRNA levels in bleomycin-treated mouse lungs and miR-101 suppression of the increase of COL1A1 and COL3A1 mRNA levels. The expression levels were relative to GAPDH. g and h, analysis of lung mechanics by Flexivent analysis. Elastance (Ers) was measured in a single-compartment model. h was measured in the Constant-Phase model. i and j, NFATc2, FZD4, FZD6, and TGFBR1 mRNA expression was increased in bleomycin-treated mouse lungs, and the increase was suppressed by miR-101 treatment as determined by real-time PCR analysis. The expression level was relative to GAPDH. n = 5 for saline + VC and saline + miR-101; n = 7–10 for bleomycin + VC and bleomycin + miR-101. The results are presented as the mean ± S.E. Statistical analyses were performed by using Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Expressing, Control, Saline, Real-time Polymerase Chain Reaction, Over Expression, Staining, Hydroxyproline Assay

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Primers for the construction of plasmids FW is forward, and RE is reverse.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Over Expression

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Identification of FZD4, FZD6, and TGFBR1 as miR-101 targets. a, 3′-UTR reporter assay: FZD4-UTR-S1 (position 1–1,432) containing miR-101-binding site 1 (position 537–543), FZD4-UTR-S2 (position 4,178–5,463) containing miR-101-binding site 2 (position 1,240–1,246), full length of FZD6-UTR, TGFBR1-UTR-S1 (position 66–1,524) containing miR-101-binding site 1 (position 460–466), or TGFBR1-UTR-S2 (position 3,210–4,749) containing miR-101-binding site 2 (position 3,993–3,999) were co-transfected into HEK 293T cells with miR-101-1. The relative luciferase activities were measured using the Dual-Luciferase® reporter assay system. n = 3. b and c, effect of overexpressing or knockdown miR-101 on the protein expression of FZD4, FZD6, and TGFBR1 in fibroblasts. LL29 cells were treated with a lentivirus expressing miR-101-1 or the virus control (VC) at an m.o.i. of 50 for 48 h (b) or CCD-8Lu fibroblasts were treated with a lentivirus expressing anti-miR-101 or the virus control (Anti-CON) at an m.o.i. of 50 for 72 h. c, Western blotting was performed to determine the protein expression of FZD4, FZD6, and TGFBR1. The protein samples used here were the same as mentioned in Fig. 7c. The GAPDH immunoblot shown in Fig. 7c was used again here. d, overexpression of miR-101 inhibits FZD4, FZD6, and TGFBR1 mRNA expression in fibroblasts. LL29 cells were treated with lentiviral miR-101 or the virus control at an m.o.i. of 50 for 48 h. The mRNA expression of FZD4, FZD6, and TGFBR1 was determined by real-time PCR and normalized to GAPDH. The results are presented as the mean ± S.E. n = 3. e, real-time PCR showing the increased mRNA levels of FZD4, FZD6, and TGFBRR1 by anti-miR-101. CCD-8Lu fibroblasts were treated with lentiviral anti-miR-101 or the virus control at an m.o.i. of 50 for 72 h. The expression levels were relative to GAPDH from two cell preparations, each performed in duplicate. f–h, mRNA expression of FZD4, FZD6, and TGFBR1 in IPF patient lungs. The mRNA levels of FZD4, FZD6, and TGFBR1 were determined by real-time PCR and normalized to β-actin. n = 8–10. The results are presented as the mean ± S.E. Statistical analyses were performed by using Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Reporter Assay, Binding Assay, Transfection, Luciferase, Knockdown, Expressing, Virus, Control, Western Blot, Over Expression, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Overexpression of FZD4, FZD6, or TGFBR1 rescues miR-101-mediated inhibition of fibroblast proliferation and activation. a–d, knockdown of TGFBR1 repressed the TGF-β1-induced mRNA expression of α-SMA, COL1A1, COL3A1, and COL4A1 in fibroblasts. LL29 cells were treated with lentivirus shRNA-TGFBR1 or the virus control at an m.o.i. of 50 for 48 h. Then, the cells were stimulated with 5 ng/ml TGF-β1 for 48 h. The α-SMA, COL1A1, COL3A1, and COL4A1 mRNA expression levels were determined by real-time PCR. The expression levels were relative to GAPDH from two cell preparations, each performed in duplicate. The results are presented as the mean ± S.D. **, p < 0.01. e and f, overexpression of FZD4 or FZD6 rescues the miR-101-mediated inhibition of fibroblast proliferation. LL29 cells were split into 96-well plates at 3,000 cells per well. After a 24-h culture, cells were infected with lentiviruses at a total of m.o.i. = 50 (miR-Con miR-101, GFP, FZD4, and FZD6 at m.o.i. = 25 each, virus control (VC) = miR-Con + GFP) for 48 h. Cells were starved for another 24 h and stimulated with WNT5a (1 μg/ml) for 12 h. FZD4 and FZD6 expression was determined by Western blotting by using anti-GFP antibody. Cell proliferation was determined by BrdU assay. Data were presented as mean ± S.E. Statistical analysis was performed by ANOVA and followed by Tukey's HSD test. n = 4. **, p < 0.01. g–j, overexpression of TGFBR1 rescues miR-101-mediated inhibition of α-SMA, COL1A1, and COL3A1 mRNA expression. LL29 cells were infected with lentiviruses at a total of m.o.i. = 50 (miR-Con, miR-101, GFP, and TGFBR1 at m.o.i. = 25 each, virus control = miR-Con + GFP) for 48 h. Cells were then stimulated with TGFβ1 (5 ng/ml) for 48 h. Cells were collected for real-time PCR. The results are presented as the mean ± S.E., n = 3. ANOVA followed by Tukey's HSD test was performed. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Over Expression, Inhibition, Activation Assay, Knockdown, Expressing, shRNA, Virus, Control, Real-time Polymerase Chain Reaction, Infection, Western Blot, BrdU Staining

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Adenovirus-mediated miR-101 gene transfer attenuates bleomycin-induced pulmonary fibrosis. On day 1, an adenovirus expressing miR-101-1 or the control adenovirus (VC) (5 × 109 pfu per mouse) was delivered into the lungs of 6–8-week-old C57BL/6 mice by nasal instillation. On day 2, bleomycin (Bleo) (0.06 units per mouse) or the same volume of saline (Sal) was intranasally instilled into the lungs. On day 15, the mice were subjected to the analysis of respiratory mechanics by Flexivent and then sacrificed. The left lungs were collected for RNA and protein analysis, and the right lungs were fixed for histological analysis. a, real-time PCR analysis showing adenovirus-mediated overexpression of miR-101 in the mouse lung. The expression levels were relative to U6. b, H&E staining showing the fibrotic changes in the mouse lung induced by bleomycin. miR-101 attenuated the fibrotic changes in bleomycin-treated mouse lungs. Scale bar, 100 μm. c, Ashcroft score grade of pulmonary fibrosis. d, lung collagen content as determined using the QuickZyme hydroxyproline assay kit. e and f, real-time PCR analysis showing the increased COL1A1 and COL3A1 mRNA levels in bleomycin-treated mouse lungs and miR-101 suppression of the increase of COL1A1 and COL3A1 mRNA levels. The expression levels were relative to GAPDH. g and h, analysis of lung mechanics by Flexivent analysis. Elastance (Ers) was measured in a single-compartment model. h was measured in the Constant-Phase model. i and j, NFATc2, FZD4, FZD6, and TGFBR1 mRNA expression was increased in bleomycin-treated mouse lungs, and the increase was suppressed by miR-101 treatment as determined by real-time PCR analysis. The expression level was relative to GAPDH. n = 5 for saline + VC and saline + miR-101; n = 7–10 for bleomycin + VC and bleomycin + miR-101. The results are presented as the mean ± S.E. Statistical analyses were performed by using Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Expressing, Control, Saline, Real-time Polymerase Chain Reaction, Over Expression, Staining, Hydroxyproline Assay

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation

doi: 10.1074/jbc.M117.805747

Figure Lengend Snippet: Primers for the construction of plasmids FW is forward, and RE is reverse.

Article Snippet: FZD4, FZD6, and TGFBR1 expression vectors The open reading frame of FZD4, FZD6, and TGFBR1 was amplified by PCR using specific primers ( ) from mouse cDNA clones purchased from OriGene (FZD4, catalogue no. MC203860; FZD6, catalogue no. MC204555; TGFBR1, catalogue no. MR20891).

Techniques: Over Expression

Journal: Oncogene

Article Title: The TβRI promotes migration and metastasis through thrombospondin 1 and ITGAV in prostate cancer cells

doi: 10.1038/s41388-024-03165-3

Figure Lengend Snippet: a Exon 2 of TβRI was designed to be targeted by the mutant Cas9 enzyme. The mutant clone A9 contained a deletion of TβRI as detected by sequencing and the stop sequence formed by a frameshift mutation. b The mutant cell clones (A–E) and wild-type (WT) cells were examined by Cel-1 assay (Surveyor nuclease assay). c Mutant clones B5 and A9 and WT cells detected by RT-PCR. d , e Western blot and f qPCR were used to verify the TβRI-deficient cell lines. Results are shown as mean ± SEM, * p < 0.05, ** p < 0.01, Student’s t -test. g WT, A9, and HA - TβR1 reconstituted A9 cells were lysed and subjected to immunoblotting with antibodies TβRI, pSmad2, Smad2, and actin. h CAGA-luciferase reporter assay showing the TGFβ response of the indicated cell lines. Results are shown as mean ± SEM, * p < 0.05, *** p < 0.001, Student’s t -test.

Article Snippet: ML is a founder, shareholder, and board member of the company MetaCurUm Biotech AB that develops TβRI-based cancer therapies and biomarkers.

Techniques: Mutagenesis, Sequencing, Clone Assay, Nuclease Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Reporter Assay

Journal: Oncogene

Article Title: The TβRI promotes migration and metastasis through thrombospondin 1 and ITGAV in prostate cancer cells

doi: 10.1038/s41388-024-03165-3

Figure Lengend Snippet: a Gene-modified cell lines wild-type (WT), A9 (TβRI deficient), A9 HA-TβRI (A9 cells reconstituted with HA-TβRI), and A9 vector (empty vector as control) were subjected to immunoblotting with antibodies pSmad2, Smad2, TβRI, and actin. b CAGA-luciferase reporter assay showing the TGFβ response in the indicated cell lines. Results are shown as mean ± SEM, *** p < 0.001, Student’s t -test. c Expression of Smad7 and SERPINE1 in the indicated cell lines detected by qPCR. Results are shown as mean ± SEM, *** p < 0.001, Student’s t -test. d Microarray data showing the differentially expressed genes (DEGs) in the indicated cell lines with or without TGFβ treatment. e DEGs were annotated to the biological functions by DAVID Bioinformatics Resources. * p < 0.05, ** p < 0.01.

Article Snippet: ML is a founder, shareholder, and board member of the company MetaCurUm Biotech AB that develops TβRI-based cancer therapies and biomarkers.

Techniques: Modification, Plasmid Preparation, Control, Western Blot, Luciferase, Reporter Assay, Expressing, Microarray

Journal: Oncogene

Article Title: The TβRI promotes migration and metastasis through thrombospondin 1 and ITGAV in prostate cancer cells

doi: 10.1038/s41388-024-03165-3

Figure Lengend Snippet: a – c Expression of THBS1 and ITGAV detected by western blotting in the PC3U, DU-145, and A549 cell lines with or without a knockout or knockdown of TβRI. d Immunofluorescent (IF) staining of THBS1 and pSmad2 in WT and A9 cells in a wound-healing assay with or without TGFβ treatment. Scale bar, 20 μm. e Invasion assay showing the invasive capacity of PC3U cells with or without knockout of TβRI or with or without TGFβ treatment for 48 h. Scale bar, 50 μm. f Western blotting showing the overexpression of THBS1 in A9 cells. g Invasion assay showing the invasive capacity of WT, A9, and A9 cells overexpressed THBS1. Scale bar, 50 μm. h The invading cells were quantified by OD value. Shown as mean ± SD, * p < 0.05. i IF staining of THBS1 and Paxillin in PC3U cells treated with TGFβ for 6 h. Scale bar, 20 μm. j IF staining of THBS1, Paxillin, and Phalloidin in PC3U cells treated with TGFβ for 6 h. Scale bar, 20 μm.

Article Snippet: ML is a founder, shareholder, and board member of the company MetaCurUm Biotech AB that develops TβRI-based cancer therapies and biomarkers.

Techniques: Expressing, Western Blot, Knock-Out, Knockdown, Staining, Wound Healing Assay, Invasion Assay, Over Expression

Journal: Oncogene

Article Title: The TβRI promotes migration and metastasis through thrombospondin 1 and ITGAV in prostate cancer cells

doi: 10.1038/s41388-024-03165-3

Figure Lengend Snippet: a Co-IF staining of HA, THBS1, and ITGAV in the wild-type (WT), A9 (TβRI deficient), A9 HA-TβRI (A9 cells reconstituted with HA-TβRI) cell lines with or without TGFβ treatment. Scale bar, 20 μm. b Co-IP was performed to detect the interaction of HA-TβRI with THBS1 and ITGAV. c The correlation of TβRI and THBS1 expression, TβRI and ITGAV expression in prostate cancer (TCGA database). d The interaction of THBS1 and ITGAV was detected in a wound-healing assay by in situ PLA. Scale bar, 50 μm. e Invasion assay showing that knockdown of THSB1 prevented the invasion capacity of PC3U cells treated with TGFβ. Scale bar, 50 μm. f The invading cells were quantified by OD value. Shown as mean ± SD, * p < 0.05. g Western blot showing the expression of THSB1 and ITGAV in PC3U cells with THSB1 knockdown.

Article Snippet: ML is a founder, shareholder, and board member of the company MetaCurUm Biotech AB that develops TβRI-based cancer therapies and biomarkers.

Techniques: Staining, Co-Immunoprecipitation Assay, Expressing, Wound Healing Assay, In Situ, Invasion Assay, Knockdown, Western Blot

Journal: Oncogene

Article Title: The TβRI promotes migration and metastasis through thrombospondin 1 and ITGAV in prostate cancer cells

doi: 10.1038/s41388-024-03165-3

Figure Lengend Snippet: a Wild-type (WT) PC3U and TβRI-deficient A9 cells were injected into the prostates of mice. Tumors and sentinel lymph nodes were obtained from the orthotopic xenograft mouse model after 4 weeks. b The tumor weight was quantified in both the WT ( n = 9) and A9 ( n = 9) groups. Results are shown as mean ± SEM, *** p < 0.001, one-way ANOVA. c The metastatic lymph nodes were quantified in both the WT ( n = 9) and A9 ( n = 6) groups. Results are shown as mean ± SEM, *** p < 0.001, one-way ANOVA. d Representative images of THBS1 staining in the WT or A9 tumors and lymph metastasis. Scale bar, 500 μm. Left: the black arrow shows the lymph vessels in the tumor-invasive front, and the red arrow shows tumor cells infiltrating into the adjacent muscles. Right: the black arrows show the remaining lymphatic cells in the sentinel lymph nodes. Scale bar, 100 μm. e Representative images of THBS1 staining in the normal adjacent prostate tissue, lower malignant prostatic adenocarcinoma (GS ≤ 6), and higher malignant prostatic adenocarcinoma (GS > 6). f THBS1 expression was examined on tissue microarray sections with 192 samples. Immunoreactivity (IR) was scored as negative (IR = 0), weak (IR = 1), moderate (IR = 2), or strong (IR = 3) **** p < 0.0001, Tukey’s multiple comparisons test. g A proteomic database was used to analyze the THBS1 protein level in the control adjacent prostate tissue ( n = 8), localized tumor ( n = 28), and bone metastasis ( n = 22). **** p < 0.0001, one-way ANOVA. h Representative images for the IF staining of THBS1 and panCK AE1/3 in the human primary and bone metastatic tumors. Scale bar, 50 μm.

Article Snippet: ML is a founder, shareholder, and board member of the company MetaCurUm Biotech AB that develops TβRI-based cancer therapies and biomarkers.

Techniques: Injection, Staining, Muscles, Expressing, Microarray, Control

Journal: Nature Communications

Article Title: Targeting hepatocytic TβRI ameliorates liver metastatic outcomes by revitalizing stem-like CD8 + Tex subsets

doi: 10.1038/s41467-025-65615-0

Figure Lengend Snippet: a IHC staining with TβRI-specific antibody to detect the distribution of TβRI + cells in colorectal liver metastases ( n = 36 patients). Representative IHC image. Scale bar, 50 μm. The bar chart in the right represents the percentage of high and low TβRI expression cases in liver metastasis, adjacent and distal liver tissues. p < 0.001 by two-sided Pearson’s chi-square test. b Overall survival curves based on adjacent liver TβRI expressed level in CRLM patients using the multivariate COX regression analysis, a two-sided P -value of < 0.05 was considered statistically significant. c Representative images depicting multicolor immunohistochemical co-staining of TβRI with specific cell type markers in CRLM adjacent liver tissues (DAPI: cell nucleus, HNF4α: hepatocytes, α-SMA: hepatic stellate cells, CD68: macrophages, PAN-CK: tumor cells). Scale bar, 25 μm. d Experimental scheme for generating TβRI flox/flox Alb-Cre +/- ( TβRI-hKO ) mice. TβRI flox/flox Alb-Cre -/- and C57BL/6 J were used as TβRI-hWT mice. e Representative bioluminescent image and Quantification of TβRI-hWT versus TβRI-hKO mice inoculated with MC38-Luc cells ( n = 6 mice/group). f Representative macroscopic image of ( e ) and Quantification of Liver to body weight ratio (LBR). g Representative bioluminescent image of TβRI-hWT versus TβRI-hKO mice inoculated with PAN02-Luc cells ( n = 5 mice/group). h Quantification of ( g ). i Representative macroscopic image of ( g ) and Quantification of LBR. j Representative bioluminescent image of TβRI-hWT versus TβRI-hKO mice inoculated with B16F10-Luc cells ( n = 5 mice/group). k Quantification of ( j ). l Representative macroscopic image of ( j ) and Quantification of LBR. Data are presented as the mean ± SD, and were analyzed with an unpaired two-sided Student’s t test ( e–l ). Consistent results were observed across three biological replicates in ( e–l ). Source data are provided as a Source Data file.

Article Snippet: The above findings prompted us to explore whether therapeutic interventions, specifically Galectin-9 neutralization (αGal-9) or TβRI inhibition (Galunisertib) , would similarly alleviate liver metastasis by reshaping CD8 + T cell responses in our mouse model.

Techniques: Immunohistochemistry, Expressing, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: Targeting hepatocytic TβRI ameliorates liver metastatic outcomes by revitalizing stem-like CD8 + Tex subsets

doi: 10.1038/s41467-025-65615-0

Figure Lengend Snippet: a , b Frequency of T cell subsets based on the expression of Ly108 and CX3CR1 within CD8 + PD1 + CD44 + tumor-reactive T cells in different anatomical locations of TβRI-hWT and TβRI-hKO mice ( n = 6 mice/group). Flow cytometry representative plots ( a ). Statistical analysis of these subsets in liver metastases across multiple tumor types are shown ( b ). Statistical analysis of these subsets in the spleen and PBMC are not shown. c–f Flow cytometry was used to detect the phenotypes, transcriptional profiles, and effector functions of Ly108 + CX3CR1 - , Ly108 + CX3CR1 + , Ly108 - CX3CR1 + , Ly108 - CX3CR1 - subsets in liver metastases of TβRI-hWT and TβRI-hKO mice inoculated with PAN02 on day 28. c Representative histograms showing the expression of PD-1, LAG3, TIGIT, TIM3, and 2B4 among these T subsets ( n = 6 mice/group). MFI analysis of TIM3 and 2B4 was summarized in the right statistical chart. d Representative flow cytometry plots and statistical analysis depicting the TOX/TCF1 co-expression among these T subsets ( n = 4 mice/group). e Representative flow cytometry plots and statistical analysis depicting the TNF and IFNγ co-production by these T subsets upon ex vivo CD3/CD28 stimulation ( n = 5 mice/group). f Representative flow cytometry plots and statistical analysis depicting the percentages of these T subsets degranulating (CD107a) and producing IL2 upon ex vivo CD3/CD28 stimulation ( n = 5 mice/group). Data are presented as the mean ± SD. Data were analyzed with unpaired two-sided Student’s t test ( b ) and two-way ANOVA with Tukey’s multiple comparisons test ( c–f ). Consistent results were observed across three biological replicates in ( a–f ). Source data are provided as a Source Data file.

Article Snippet: The above findings prompted us to explore whether therapeutic interventions, specifically Galectin-9 neutralization (αGal-9) or TβRI inhibition (Galunisertib) , would similarly alleviate liver metastasis by reshaping CD8 + T cell responses in our mouse model.

Techniques: Expressing, Flow Cytometry, Ex Vivo

Journal: Nature Communications

Article Title: Targeting hepatocytic TβRI ameliorates liver metastatic outcomes by revitalizing stem-like CD8 + Tex subsets

doi: 10.1038/s41467-025-65615-0

Figure Lengend Snippet: a Experimental design. b Representative bioluminescent image of pre-adoptive and post-adoptive C57BL/6 J mice receiving Ly108 + CX3CR1 - , Ly108 + CX3CR1 + , Ly108 - CX3CR1 + , Ly108 - CX3CR1 - cells transfer or without transfer ( n = 5 mice/group). c Quantification of ( b ). d Survival curve of tumor-bearing mice for indicated groups described in ( b ). Log-rank test for survival comparison. e Flow cytometry depicted the phenotype of adoptively transferred cells before and 14 days after transfer. All subsets shown were gated on CD8 + PD1 + CD44 + CD45.2 + CD45.1 - cells. f Representative bioluminescent image of TβRI-hWT and TβRI-hKO mice inoculated with TGFβ1 OE , TGFβ1 KD , or TGFβ1 CTL PAN02-Luc cells ( n = 6 mice/group). g Quantification of ( f ). Data are presented as the mean ± SD, and were analyzed with one-way ( g ) or two-way ( c ) ANOVA with Tukey’s multiple comparison test. Consistent results were observed across three biological replicates in ( b–g ). Source data are provided as a Source Data file.

Article Snippet: The above findings prompted us to explore whether therapeutic interventions, specifically Galectin-9 neutralization (αGal-9) or TβRI inhibition (Galunisertib) , would similarly alleviate liver metastasis by reshaping CD8 + T cell responses in our mouse model.

Techniques: Comparison, Flow Cytometry

Journal: Nature Communications

Article Title: Targeting hepatocytic TβRI ameliorates liver metastatic outcomes by revitalizing stem-like CD8 + Tex subsets

doi: 10.1038/s41467-025-65615-0

Figure Lengend Snippet: a Experimental setup to assess the indispensable role of TAMs or CD8 + T cells in improving metastatic outcomes of PAN02-bearing mice resulting from hepatocytic-TβRI ablation. b The M1-to-M2 ratio. c Representative bioluminescent image of TβRI-hWT and TβRI-hKO mice treated with vehicle (veh), Clodronate (CLD) liposome or αCD8 (n = 5 mice/group). d Survival curve of tumor-bearing mice for the indicated groups described in ( c ). Log-rank test for survival comparison. e Quantification of ( c ). f Representative flow cytometry plots and Statistical analysis depicting the percentages of Tex subsets in liver metastases of veh or CLD liposome-treated TβRI-hWT and TβRI-hKO mice on days 28. g Representative bioluminescent image and liver macroscopic image of TβRI-hWT and TβRI-hKO mice inoculated with PAN02 + BMDM or PAN02 alone ( n = 5 mice/group). h Quantification of ( g ). i Statistical analysis depicting the percentages of Tex subsets about the indicated groups. Data are presented as the mean ± SD, and were analyzed with one-way ( h ) or two-way ( b , e , f , i ) ANOVA with Tukey’s multiple comparison test. Consistent results were observed across three biological replicates in ( b–i ). Source data are provided as a Source Data file.

Article Snippet: The above findings prompted us to explore whether therapeutic interventions, specifically Galectin-9 neutralization (αGal-9) or TβRI inhibition (Galunisertib) , would similarly alleviate liver metastasis by reshaping CD8 + T cell responses in our mouse model.

Techniques: Comparison, Flow Cytometry

Journal: Nature Communications

Article Title: Targeting hepatocytic TβRI ameliorates liver metastatic outcomes by revitalizing stem-like CD8 + Tex subsets

doi: 10.1038/s41467-025-65615-0

Figure Lengend Snippet: a Luminex showing the protein levels of inflammatory cytokines in tumor interstitial fluid of TβRI-hWT and TβRI-hKO mice ( n = 3 mice/group). Data are shown as a heatmap. b Experimental scheme for generating Gal-9 flox/flox Alb-Cre +/- (Gal9-hKO) mice. Gal-9 flox/flox Alb-Cre -/- and C57BL/6 J were used as Gal9-hWT mice. c Representative bioluminescent image of Gal9-hWT versus Gal9-hKO mice inoculated with TGFβ1 OE or TGFβ1 CTL -PAN02 cells in the portal vein on day 28 ( n = 6 mice/group). d Bioluminescence quantification of ( c ). e Statistical analysis depicting the percentages of CD39 + TOX + cells within CD8 + PD1 + CD44 + tumor-reactive T cells in the indicated groups. f Statistical analysis depicting the percentages of Tex subsets based on TCF1 and CX3CR1 within CD8 + PD1 + CD44 + tumor-reactive T cells. g statistical analysis depicting the TNF and IFNγ co-production by CD8 + CD44 + T subsets in the indicated groups upon ex vivo CD3/CD28 stimulation. h The diagram of experimental design. i Representative bioluminescent image of Gal9-hWT versus Gal9-hKO mice treated with IgG, αCD4, αCD8 or αPD-1( n = 5 mice/group). j Representative macroscopic image of ( i ). k Bioluminescence quantification of ( i ). l Statistical analysis depicting the percentages of Tex subsets based on Ly108 and CX3CR1 within CD8 + PD1 + CD44 + tumor-reactive T cells. Data are presented as the mean ± SD, and were analyzed with unpaired two-sided Student’s t test ( k ) or two-way ( d , e , f , g , l ) ANOVA with Tukey’s multiple comparison test. Consistent results were observed across three biological replicates in ( a , c–g , i–l ). Source data are provided as a Source Data file.

Article Snippet: The above findings prompted us to explore whether therapeutic interventions, specifically Galectin-9 neutralization (αGal-9) or TβRI inhibition (Galunisertib) , would similarly alleviate liver metastasis by reshaping CD8 + T cell responses in our mouse model.

Techniques: Luminex, Ex Vivo, Comparison

Journal: Nature Communications

Article Title: Targeting hepatocytic TβRI ameliorates liver metastatic outcomes by revitalizing stem-like CD8 + Tex subsets

doi: 10.1038/s41467-025-65615-0

Figure Lengend Snippet: a Experimental setup to assess the effects of αGal-9 or Galunisertib monotherapy and combination therapy in improving outcomes in B16F10-bearing C57BL/6 J mice with liver metastases. ( n = 6 mice, Vehicle + IgG; n = 6 mice, Galunisertib + IgG; n = 6 mice, Vehicle + αGal-9, n = 5 mice, Galunisertib + αGal-9). b Representative bioluminescent and macroscopic image of B16F10-bearing C57BL/6 J mice for indicated groups describe in ( a ). c Quantification of bioluminescence and LBR. d Survival curve of tumor-bearing mice for the indicated groups described in ( b ). Log-rank test for survival comparison. e Representative flow cytometry plots depicting the percentages of Tex subsets based on TCF1and CX3CR1. f Statistical analysis of ( e ). g Representative flow cytometry plots depicting the percentages of Tex subsets based on TOX/CX3CR1. h Statistical analysis of ( g ). i Representative flow cytometry plots based depicting the percentages of Tex subsets on Ki-67 and TCF1. j Statistical analysis of ( i ). k–m Representative flow cytometry plots ( k ) and statistical analysis ( l , m ) depicting the TNF and IFNγ co-production by CD8 + T cells upon ex vivo CD3/CD28 stimulation for the indicated groups. Data are presented as the mean ± SD, and were analyzed with one-way ( c , l , m ) or two-way ( f , h , g ) ANOVA with Sidak’s multiple comparison test. Consistent results were observed across three biological replicates in ( b–m ). Source data are provided as a Source Data file.

Article Snippet: The above findings prompted us to explore whether therapeutic interventions, specifically Galectin-9 neutralization (αGal-9) or TβRI inhibition (Galunisertib) , would similarly alleviate liver metastasis by reshaping CD8 + T cell responses in our mouse model.

Techniques: Comparison, Flow Cytometry, Ex Vivo