Journal: Frontiers in Immunology

Article Title: PTN/IGF-2 signaling modulates endometrial decidualization and immune cell trafficking to facilitate pregnancy maintenance

doi: 10.3389/fimmu.2026.1790942

Figure Lengend Snippet: PTN regulates endometrial stromal cell decidualization through the IGF-2 signaling axis. (A) PPI network of PTN, IGF-2, IGFBP1, LIF, PRL, and other decidualization-related genes. (B) The expression levels of PTN and its interacting partners (IGF-2, WNT4, PRL, IGFBP1, and LIF) in the RIF (left) and RPL (right) datasets. (C) Expressions of decidualization-related genes (including IGFBP1 , LIF , PRL , and WNT4 ) in NC or si PTN hESCs after treatment with control vehicle, cAMP, and IGF-2 via RT-qPCR (n = 9). (D) Immunoblotting for PTN, IGFBP1, and PRL expression levels with control vehicle, cAMP, and IGF-2. (E) ELISA for IGF-2 levels with control vehicle, cAMP, and IGF-2. (F) Schematic diagram of the experiments performed using the different treatments of hESCs. Data are presented as mean ± SEM and analyzed using t-test or one-way ANOVA test. * compared with NC treated with control vehicle, # compared with si PTN treated with control vehicle, $ compared with si PTN treated with control cAMP (NS, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ####p < 0.0001, $p < 0.05, $$p < 0.01, $$$p < 0.001, $$$$p< 0.0001). PPI, protein–protein interaction; RIF, recurrent implantation failure; RPL, recurrent pregnancy loss; hESCs, human endometrial stromal cells; cAMP, cyclic adenosine monophosphate.

Article Snippet: The remaining cells were digested with trypsin and inoculated into a new culture dish, and they were treated with control vehicle or IGF-2 (50 ng/mL, R&D Systems, Minneapolis, MN, USA, 292-G2) for 48 h and then collected for quantitative real-time polymerase chain reaction (qRT-PCR).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: PTN/IGF-2 signaling modulates endometrial decidualization and immune cell trafficking to facilitate pregnancy maintenance

doi: 10.3389/fimmu.2026.1790942

Figure Lengend Snippet: IGF-2 promotes decidualization and rescues decidualization defects caused by PTN deficiency. (A) Schematic diagram of the experiments performed using the different treatments of ESCs isolated from endometrium of RIF and RPL patients. (B, C) RT-qPCR analysis of levels of decidualization-related genes in ESCs isolated from endometrium of RIF (B) and RPL (C) patients after IGF-2 (50 ng/mL) treatment for 48 h (n = 9). (D) The flowchart depicts the steps involved in establishing a mouse model of intrauterine perfusion with siRNA-mediated knockdown of PTN and then supplemented with IGF-2 (50 ng/mL) through tail vein injection. (E) Expression of PTN after Ptn knockdown and supplemented with control vehicle or IGF-2 (50 ng/mL) in the mouse endometrium, measured by immunofluorescence, and quantitative analysis of immunofluorescence was performed (n = 6). (F) RT-qPCR analysis of levels of decidualization-related genes after Ptn knockdown and supplemented with control vehicle or IGF-2 (50 ng/mL) in the mouse endometrium (n = 6). Data are presented as mean ± SEM and analyzed using t-test or one-way ANOVA test. * compared with NC treated with control vehicle, # compared with si PTN treated with control vehicle (NS, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001, ## p < 0.01, ### p < 0.001, #### p < 0.0001). ESCs, endometrial stromal cells; PPI, protein–protein interaction; RIF, recurrent implantation failure.

Article Snippet: The remaining cells were digested with trypsin and inoculated into a new culture dish, and they were treated with control vehicle or IGF-2 (50 ng/mL, R&D Systems, Minneapolis, MN, USA, 292-G2) for 48 h and then collected for quantitative real-time polymerase chain reaction (qRT-PCR).

Techniques: Isolation, Quantitative RT-PCR, Knockdown, Injection, Expressing, Control, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: PTN/IGF-2 signaling modulates endometrial decidualization and immune cell trafficking to facilitate pregnancy maintenance

doi: 10.3389/fimmu.2026.1790942

Figure Lengend Snippet: PTN deficiency induces endometrial immune imbalance and leads to adverse pregnancy outcomes, which can be partially reversed by IGF-2. (A) PPI network of PTN, IGF-2, CXCR4, CD4, CD8, CD56, and other immunoregulators. (B, C) Flow cytometry analysis (B) and statistical quantification (C) of cell populations of NK cells and T cells, and the expression levels of CD16, CXCR4, and GZMB in NK cell form the endometrial tissues of NC and si PTN mice treated with control vehicle or IGF-2 (50 ng/mL) (n = 6). (D) The flowchart depicts the steps involved in establishing a mouse model of intrauterine perfusion with siRNA-mediated knockdown of Ptn and then supplemented with IGF-2 (50 ng/mL) through tail vein injection for 2 days; then, these female mice were mated with fertile male mice; analysis of fertility, including pregnancy rate, IF, and fluorescence-activated cell sorting (FACS). were performed at gestational day 13.5. (E) Representative images of uteri from pregnant mice in the NC and si PTN mice treated with control vehicle or IGF-2 (50 ng/mL) at gestational day 13.5 (n = 6) (arrow shows the absorption site). (F) Pregnancy rate (%) in NC and si PTN mice treated with control vehicle or IGF-2 (50 ng/mL) (n = 15). (G) Quantification of embryo numbers (left) and absorption rate (right) per mouse in NC (n = 13) and si PTN mice treated with control vehicle (n = 6) or IGF-2 (50 ng/mL) (n = 11) at gestational day 13.5. Data are presented as mean ± SEM and analyzed using one-way ANOVA test or χ 2 test. * Compared with NC treated with control vehicle, # compared with si PTN treated with control vehicle (NS, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ## p < 0.01, ### p < 0.001, #### p < 0.0001). IF, immunofluorescence.

Article Snippet: The remaining cells were digested with trypsin and inoculated into a new culture dish, and they were treated with control vehicle or IGF-2 (50 ng/mL, R&D Systems, Minneapolis, MN, USA, 292-G2) for 48 h and then collected for quantitative real-time polymerase chain reaction (qRT-PCR).

Techniques: Flow Cytometry, Expressing, Control, Knockdown, Injection, Fluorescence, FACS, Immunofluorescence

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex-mediated stimulation on INSR/IGF1R and ERK1/2. HEK293 cells were pretreated with dimethyl sulfoxide (linsitinib [−]) or with linsitinib (linsitinib [+]) and subsequently stimulated with Pas2r12, Pas2r12–EGFP, Pas2r12–IgG, or insulin for 2 min ( A – C ) or 10 min ( D – F ). Panels A and D show representative Western blot images. Panels B and E display the phosphorylation levels of INSR/IGF1R (pINSR/pIGF1R); panels C and F show the phosphorylation levels of ERK1/2 (pERK1/2); all values were normalized to GAPDH. Phosphorylation levels were analyzed and compared with the solvent control (DMEM only, linsitinib [−]) using Student’s t -test. For each treatment condition, linsitinib (+) was compared with the corresponding linsitinib (−) condition. Statistically significant differences compared with the control are indicated on the bar graphs; significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Statistical comparisons were performed against control cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05 and ** p < 0.01. N = 4.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Western Blot, Phospho-proteomics, Solvent, Control

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effect of IGF1R and INSR knockdown on the Pas2r12-mediated cytosolic delivery of EGFP. Western blot analyses ( A – C ) and confocal laser scanning microscopy images ( D , E ). Graphs B and C show IGF1R and INSR expression levels, respectively, normalized to GAPDH. Cells analyzed with confocal laser scanning microscopy in panel A were analyzed by Western blot ( A – C ). ( D ) Pas2r12-mediated cytosolic delivery of EGFP in knockdown cells, with EGFP fluorescence shown in green, and ( E ) percentage of cells exhibiting cytosolic EGFP delivery. Scale bars represent 20 μm. Statistical comparisons were performed against siNC cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 4.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Knockdown, Western Blot, Confocal Laser Scanning Microscopy, Expressing, Fluorescence

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Assessment of IGF1R overexpression. ( A ) Verification of IGF1R expression levels in HEKI cells. ( B ) Relative levels of IGF1R expression normalized to GAPDH based on the data in panel A. Values for IGF1R/GAPDH are expressed compared with parental HEK293 cells (set to 1). Statistical comparisons were performed against HEK293 cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 3. ( C ) Subcellular localization of IGF1R is shown in green; nuclei were counterstained with Hoechst 33342 (blue). Scale bars represent 20 μm.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Over Expression, Expressing

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex stimulation on INSR/IGF1R in IGF1R-overexpressing cells. ( A ) HEKI#66 cells pretreated with DMSO (linsitinib [−]) or linsitinib (linsitinib [+]) were stimulated with Pas2r12, Pas2r12–EGFP, or insulin for 2 min. Level of phosphorylated INSR/IGF1R (pINSR/pIGF1R) was assessed by Western blot. ( B ) Relative levels of pINSR/pIGF1R, normalized to GAPDH, corresponding to the data in panel A. Phosphorylation levels were analyzed using Student’s t -test. Each treatment condition was compared with the solvent-treated HEKI#66 control (linsitinib [−]), and, for each treatment, phosphorylation levels in the linsitinib (−) and linsitinib (+) conditions were also compared. Statistically significant differences compared with the solvent-treated control are indicated on the bar graphs, and significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 3.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Western Blot, Phospho-proteomics, Solvent, Control

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effect of IGF1R overexpression on the Pas2r12-mediated cytosolic delivery of EGFP. ( A ) Representative confocal images showing the cellular uptake of Pas2r12–EGFP in IGF1R-overexpressing (HEKI) cells. Scale bars represent 20 μm. ( B ) Relative levels of the cytosolic delivery efficiency of EGFP in HEKI cells compared with parental HEK293 cells (set to 100%). Merged images show EGFP fluorescence (green) and differential interference contrast. Statistical comparisons were performed against HEK293 cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). ** p < 0.01. N = 3.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Over Expression, Fluorescence

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex stimulation on INSR/IGF1R in INSR-overexpressing cells. ( A ) IN#1 cells pretreated with DMSO (linsitinib [−]) or linsitinib (linsitinib [+]) were stimulated with Pas2r12, Pas2r12–EGFP, or insulin for 2 min. Phosphorylation of INSR/IGF1R (pINSR/pIGF1R) was assessed by Western blot. ( B ) Relative levels of pINSR/pIGF1R were normalized to GAPDH, corresponding to the data in panel A. Phosphorylation levels were analyzed using Student’s t -test. Each treatment condition was compared with the solvent-treated IN#1 control (linsitinib [−]), and for each treatment, phosphorylation levels in the linsitinib (−) and linsitinib (+) conditions were also compared. Statistically significant differences compared with the solvent-treated control are indicated on the bar graphs, and significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Error bars indicate the standard error of the mean (SEM). * p < 0.05, ** p < 0.01, and *** p < 0.001. N = 3.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Western Blot, Solvent, Control