sj-pdf-3-sci-10.1177_00368504231220765 - Supplemental material for A puzzling case report of well-differentiated neuroendocrine tumor mixed with gastric adenocarcinoma of the fundic gland type associated with autoimmune gastritis

Supplemental material, sj-pdf-3-sci-10.1177_00368504231220765 for A puzzling case report of well-differentiated neuroendocrine tumor mixed with gastric adenocarcinoma of the fundic gland type associated with autoimmune gastritis by Zheng Wang, Weixun Zhou, Jingnan Li, Wenlong Wen, Zhiyong Liang and Zhen Huo in Science Progress</p

sj-pdf-2-sci-10.1177_00368504231220765 - Supplemental material for A puzzling case report of well-differentiated neuroendocrine tumor mixed with gastric adenocarcinoma of the fundic gland type associated with autoimmune gastritis

Supplemental material, sj-pdf-2-sci-10.1177_00368504231220765 for A puzzling case report of well-differentiated neuroendocrine tumor mixed with gastric adenocarcinoma of the fundic gland type associated with autoimmune gastritis by Zheng Wang, Weixun Zhou, Jingnan Li, Wenlong Wen, Zhiyong Liang and Zhen Huo in Science Progress</p

mTOR was activated during the process of pulmonary fibrosis in vivo and vitro.

<p>A) mTOR activation in fibroblast foci of lung tissue in IPF patients. a,b, H&E staining with normal control and IPF lung tissues; Immunohistochemical staining performed with α-SMA (c,d) and p-S6 (e,f) antibodies showed an increase in α-SMA and p-S6 in IPF lung tissues (d, f) compared with the control (c, e). Scale bar = 100 μm. B) mTOR activation in the lung tissues of C57BL/6J mice after bleomycin intra-tracheal injection. a,b, H&E staining with lungs of saline and bleomycin-treated mice; Immunohistochemical staining was performed with α-SMA (c,d) and p-S6 (e,f) antibodies in saline- and bleomycin-treated mouse lung tissues. NS, normal saline. Scale bar = 100 μm. C) mTOR signaling pathway was activated in primary lung fibroblasts isolated from normal controls treated with TGF-β1(5 ng/ml) for 48 h. Western blot analysis of α-SMA and p-S6 in control and TGF-β1-treated primary lung fibroblasts (a). Densitometric quantification of the Western blot in (a) is shown in (b) with α-SMA normalized against GAPDH and (c) with p-S6 normalized against S6. **, P<0.01; *, P<0.05. n = 3. D) mTOR signaling pathway was activated in MRC5 cells (a human fetal lung fibroblast cell line) treated with TGF-β1 (5 ng/ml) for 48 h. Western blot analysis of α-SMA and p-S6 in control and TGF-β1-treated MRC5 cells (a). Densitometric quantification of the Western blot in (a) is shown in (b) with α-SMA normalized against β-actin and (c) with p-S6 normalized against S6. **, p<0.01; *, p<0.05. n = 3.</p

Rapamycin-induced autophagyin the bleomycin-mediated lung injury and fibrosis model.

<p>A) Rapamycin decreased the death caused by bleomycin. Chloroquine, an autophagy inhibitor, reversed the benefit of rapamycin in the bleomycin-mediated lung injury model (Bleo+Rapa+CQ vs Bleo+Rapa, p = 0.0158). B) Western blot analysis of p62 and p-S6 were performed in the bleomycin-mediated lung injury and fibrosis model. p62, a protein inversely correlated with autophagy activity, was decreased in lungs of mice treated with rapamycin alone. p62 expression was higher with combined rapamycin and chloroquine treatment than with rapamycin alone. S6 and β-actin were used as controls. C) Western blot ananlysis of LC3 I and LC3 II were performed in the bleomycin-mediated lung injury and fibrosis mice model. D) Relative density of LC3 II/LC3 I of bands in Fig 5C. Autophagy was significantly decreased in bleomycin-mediated lung injury and fibrosis model (*bleomycin vs normal saline, p < 0.05). E) Electron microscope images of lung tissues show autophagosomes in the bleomycin-mediated lung injury model. Arrows indicate autophagosomes. Rapamycin treatment alone induced an increased number of autophagosomes. Left panel, original magnification: 6,000X and right panel, original magnification: 11,500X. F) Statistical results for the autophagosomes in Fig 5E. The statistical results indicate the percent area of autophagosomes in a cell.</p

Bleomycin-mediated lung injury in wild-type C57BL/6J mice was attenuated by rapamycin (treatment initiated at 5 days before bleomycin injection).

<p>A) H&E staining (a-d) and Masson’s trichrome staining (e-h) of mouse lungs were performed after bleomycin injection at day 21. Lung injury was milder in rapamycin-treated mice (d, h) compared with vehicle-treated mice (c, g). Scale bar = 100 μm. NS, saline; Bleo, bleomycin; Vehi, vehicle; Rapa, rapamycin. B) Semi-quantitative assessment was performed on day 21 using Ashcroft scoring method, a significantly higher score was observed in the mice treated with Bleo (no rapamycin) than those treated with Bleo+Rapa. Results were expressed as mean±SEM, n = 6 mice per group, ** p<0.01. C) Bleomycin-mediated mouse mortality was decreased after rapamycin treatment.</p

Conditional <i>Tsc1</i> knock-down in lung alveolar epithelial cells from doxycyline-treated STT mice exacerbated bleomycin-mediated lung injury.

<p>A) Histological analysis of lungs in the mice treated with bleomycin at day 21. H&E staining (a-c) and Masson’s trichrome staining (d-f) were performed. STT mice had more severe lung injury (c, f) than control mice (b, e). Scale bar = 100 μm. NS, saline; Bleo, bleomycin. B) Semi-quantitative assessment was performed on day 21 using Ashcroft scoring method, a significantly higher score was observed in STT mice treated with Bleo than control mice treated with Bleo. Results were expressed as mean±SEM, n = 6 mice per group, * p<0.05. C) STT mice had a higher mortality rate than control mice after a single intra-tracheal injection of bleomycin for 21 days.</p