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CD96-deficient mice were more resistant to IMQ-induced psoriasis-like derma...
Published: 15 December 2022
FIGURE 1. CD96-deficient mice were more resistant to IMQ-induced psoriasis-like dermatitis than were WT mice. Wild-type (WT) mice (n = 4) or Cd96−/− mice (n = 4) were treated once a day with IMQ or petroleum jelly on the shaved dorsal skin for 4 d. ( A ) Phenotypical presentation of IMQ-treated mouse dorsal skin on day 2. ( B ) Changes in erythema, scaling, and thickening of dorsal skin. ( C ) Representative histology (H&E staining) and epidermal thickness of dorsal skin on day 3. ( D ) Numbers of neutrophils (CD45+, CD11b+, Ly6G+) in the skin (1 cm2) on day 3. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Data are representative from two independent experiments (B) or two pooled experiments (C and D). FIGURE 1. CD96-deficient mice were more resistant to IMQ-induced psoriasis-like dermatitis than were WT mice. Wild-type (WT) mice (n = 4) or Cd96−/− mice (n = 4) were treated once a day with IMQ or petroleum jelly on the shaved dorsal skin for 4 d. (A) Phenotypical presentation of IMQ-treated mouse dorsal skin on day 2. (B) Changes in erythema, scaling, and thickening of dorsal skin. (C) Representative histology (H&amp;E staining) and epidermal thickness of dorsal skin on day 3. (D) Numbers of neutrophils (CD45+, CD11b+, Ly6G+) in the skin (1 cm2) on day 3. Error bars indicate SEM. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Data are representative from two independent experiments (B) or two pooled experiments (C and D). More
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CD96 on dermal γδ T cells is involved in IMQ-induced psoriasis-like dermati...
Published: 15 December 2022
FIGURE 2. CD96 on dermal γδ T cells is involved in IMQ-induced psoriasis-like dermatitis. ( A ) CD96 expression on lymphocytes and myeloid cells in the skin from naive WT or Cd96−/− mice. DC, dendritic cell; ILC, innate lymphoid cell; LC, Langerhans cell. ( B – D ) Bone marrow (BM) chimeric mice were generated by reconstituting irradiated Ly5.1+ WT mice with BM cells from Ly5.2+ WT (n = 6) or Cd96−/− (n = 6) mice. (B) Representative plot showing the chimerism of dermal (TCRδ+Vγ5) and epidermal (TCRδ+Vγ5+) γδ T cells 12 wk after BM transplantation. (C) Changes in erythema and skin thickening in BM chimeric mice treated once a day with IMQ or petroleum jelly on the shaved dorsal skin for 5 d. (D) Representative histology (H&E staining) and epidermal thickness of dorsal skin. ( E ) WT mice were treated once a day with IMQ or petroleum jelly (control) on the shaved dorsal skin for 3 d. The IL-17A–producing cell numbers/cm2 of dermal γδ T cells, epidermal γδ T cells, and CD4+ T cells (n = 4) are shown. ( F – H ) Vγ4+ γδ T cells isolated from the spleen of WT or CD96−/− mice were injected into the shaved dorsal skin of Rag1−/− mice. (F) From the next day, IMQ was applied once a day to the shaved dorsal skin for 4 d. (G) Changes in erythema and scaling of dorsal skin. (H) Representative histology (H&E staining) and the epidermal thickness of dorsal skin after IMQ treatment for 4 d (control, n = 3; PBS+IMQ, n = 4; WT γδ T+IMQ, n = 11; CD96−/− γδ T+IMQ, n = 8). Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Data are pooled from four experiments (G and H). FIGURE 2. CD96 on dermal γδ T cells is involved in IMQ-induced psoriasis-like dermatitis. (A) CD96 expression on lymphocytes and myeloid cells in the skin from naive WT or Cd96−/− mice. DC, dendritic cell; ILC, innate lymphoid cell; LC, Langerhans cell. (B–D) Bone marrow (BM) chimeric mice were generated by reconstituting irradiated Ly5.1+ WT mice with BM cells from Ly5.2+ WT (n = 6) or Cd96−/− (n = 6) mice. (B) Representative plot showing the chimerism of dermal (TCRδ+Vγ5−) and epidermal (TCRδ+Vγ5+) γδ T cells 12 wk after BM transplantation. (C) Changes in erythema and skin thickening in BM chimeric mice treated once a day with IMQ or petroleum jelly on the shaved dorsal skin for 5 d. (D) Representative histology (H&amp;E staining) and epidermal thickness of dorsal skin. (E) WT mice were treated once a day with IMQ or petroleum jelly (control) on the shaved dorsal skin for 3 d. The IL-17A–producing cell numbers/cm2 of dermal γδ T cells, epidermal γδ T cells, and CD4+ T cells (n = 4) are shown. (F–H) Vγ4+ γδ T cells isolated from the spleen of WT or CD96−/− mice were injected into the shaved dorsal skin of Rag1−/− mice. (F) From the next day, IMQ was applied once a day to the shaved dorsal skin for 4 d. (G) Changes in erythema and scaling of dorsal skin. (H) Representative histology (H&amp;E staining) and the epidermal thickness of dorsal skin after IMQ treatment for 4 d (control, n = 3; PBS+IMQ, n = 4; WT γδ T+IMQ, n = 11; CD96−/− γδ T+IMQ, n = 8). Error bars indicate SEM. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Data are pooled from four experiments (G and H). More
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CD96 on dermal γδ T cells augments IL-17A expression after IMQ treatment. (...
Published: 15 December 2022
FIGURE 3. CD96 on dermal γδ T cells augments IL-17A expression after IMQ treatment. ( A – D ) WT or Cd96−/− mice were treated once a day with IMQ or petroleum jelly (control) on the shaved dorsal skin for 3 d. (A) Il17a mRNA levels of dermal γδ T cells in the skin of WT mice (IMQ, n = 4; control, n = 3) and Cd96−/− mice (IMQ, n = 4; control, n = 3). (B and C) The proportions and the numbers of IL-17A–producing dermal γδ T cells in (B) the skin (1 cm2) and (C) the draining lymph nodes (dLNs) of WT mice (IMQ, n = 8; control, n = 6) and Cd96−/− mice (IMQ, n = 8; control, n = 6). (D) The mean fluorescence intensity (MFI) of CD69 expression on γδ T cells in dLNs from WT mice (IMQ, n = 3; control, n = 3) and Cd96−/− mice (IMQ, n = 3; control, n = 3) 3 h after IMQ treatment. The MFI of CD25 expression on γδ T cells in dLNs from WT mice (IMQ, n = 4; control, n = 3) and Cd96−/− mice (IMQ, n = 3; control, n = 3) after IMQ treatment for 3 d is shown. ( E ) BM chimeric mice were generated by reconstituting irradiated Ly5.1+Ly5.2+ mice with a 1:1 mixture of BM cells from Ly5.1+ WT and Ly5.2+Cd96−/−mice. ( F ) BM chimeric mice 12 wk after BM transplant were treated once a day with IMQ or petroleum jelly (control) on the shaved dorsal skin for 3 d. Representative plot and ratio of donor-derived Ly5.1+ WT and Ly5.2+Cd96−/− γδ T cells in the dLNs (IMQ, n = 10; control, n = 3). ( G ) Il17a mRNA levels of Ly5.1+ WT and Ly5.2+Cd96−/− γδ T cells of dLNs (IMQ, n = 10; control, n = 3). Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001. nd, not detected; ns, not significant. Data are representative from two independent experiments (A–C) or pooled from two experiments (F and G). FIGURE 3. CD96 on dermal γδ T cells augments IL-17A expression after IMQ treatment. (A–D) WT or Cd96−/− mice were treated once a day with IMQ or petroleum jelly (control) on the shaved dorsal skin for 3 d. (A) Il17a mRNA levels of dermal γδ T cells in the skin of WT mice (IMQ, n = 4; control, n = 3) and Cd96−/− mice (IMQ, n = 4; control, n = 3). (B and C) The proportions and the numbers of IL-17A–producing dermal γδ T cells in (B) the skin (1 cm2) and (C) the draining lymph nodes (dLNs) of WT mice (IMQ, n = 8; control, n = 6) and Cd96−/− mice (IMQ, n = 8; control, n = 6). (D) The mean fluorescence intensity (MFI) of CD69 expression on γδ T cells in dLNs from WT mice (IMQ, n = 3; control, n = 3) and Cd96−/− mice (IMQ, n = 3; control, n = 3) 3 h after IMQ treatment. The MFI of CD25 expression on γδ T cells in dLNs from WT mice (IMQ, n = 4; control, n = 3) and Cd96−/− mice (IMQ, n = 3; control, n = 3) after IMQ treatment for 3 d is shown. (E) BM chimeric mice were generated by reconstituting irradiated Ly5.1+Ly5.2+ mice with a 1:1 mixture of BM cells from Ly5.1+ WT and Ly5.2+ Cd96−/−mice. (F) BM chimeric mice 12 wk after BM transplant were treated once a day with IMQ or petroleum jelly (control) on the shaved dorsal skin for 3 d. Representative plot and ratio of donor-derived Ly5.1+ WT and Ly5.2+ Cd96−/− γδ T cells in the dLNs (IMQ, n = 10; control, n = 3). (G) Il17a mRNA levels of Ly5.1+ WT and Ly5.2+ Cd96−/− γδ T cells of dLNs (IMQ, n = 10; control, n = 3). Error bars indicate SEM. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. nd, not detected; ns, not significant. Data are representative from two independent experiments (A–C) or pooled from two experiments (F and G). More
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CD96 mediates a costimulatory signal in γδ T cells. ( A  and  B ) Splenic γ...
Published: 15 December 2022
FIGURE 4. CD96 mediates a costimulatory signal in γδ T cells. ( A and B ) Splenic γδ T cells of WT mice were stimulated with or without anti-CD3 mAb in combination with or without anti-CD96 mAb and/or mouse IL-23 for 18 h. (A) MFI of CD69 and CD25 expressions (n = 3 in each column). (B) Quantitative real-time PCR analysis of Il17a mRNA levels (n = 3 in each column). ( C and D ) Dermal γδ T cells of WT mice were isolated and cultured in the presence of IL-2, IL-6, IL-7, and IL-15 for 4 d and stimulated with or without anti-CD3 mAb, anti-CD96 mAb, and/or mouse IL-23 for 18 h. (C) MFI of CD69 expression (n = 3 in each column) and CD25 expression (n = 4 in each column). (D) Il17a mRNA levels (n = 4 in each column). ( E ) Splenic γδ T cells of WT mice were stimulated with anti-CD3 mAb and either anti-CD96 mAb or mouse IgG2b (cIg) and analyzed for the expression of p-ERK. Representative flow cytometry analysis data and the relative expression of p-ERK (n = 3 in each group) are shown. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. Data are representative from two independent experiments. FIGURE 4. CD96 mediates a costimulatory signal in γδ T cells. (A and B) Splenic γδ T cells of WT mice were stimulated with or without anti-CD3 mAb in combination with or without anti-CD96 mAb and/or mouse IL-23 for 18 h. (A) MFI of CD69 and CD25 expressions (n = 3 in each column). (B) Quantitative real-time PCR analysis of Il17a mRNA levels (n = 3 in each column). (C and D) Dermal γδ T cells of WT mice were isolated and cultured in the presence of IL-2, IL-6, IL-7, and IL-15 for 4 d and stimulated with or without anti-CD3 mAb, anti-CD96 mAb, and/or mouse IL-23 for 18 h. (C) MFI of CD69 expression (n = 3 in each column) and CD25 expression (n = 4 in each column). (D) Il17a mRNA levels (n = 4 in each column). (E) Splenic γδ T cells of WT mice were stimulated with anti-CD3 mAb and either anti-CD96 mAb or mouse IgG2b (cIg) and analyzed for the expression of p-ERK. Representative flow cytometry analysis data and the relative expression of p-ERK (n = 3 in each group) are shown. Error bars indicate SEM. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. ns, not significant. Data are representative from two independent experiments. More
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Blockade of CD96 ameliorates IMQ-induced psoriasis-like dermatitis. WT or ...
Published: 15 December 2022
FIGURE 5. Blockade of CD96 ameliorates IMQ-induced psoriasis-like dermatitis. WT or Cd96−/− mice were treated once a day with IMQ or petroleum jelly on the shaved dorsal skin for 3 d. ( A ) Quantitative real-time PCR analysis of Cd96 mRNA levels of dermal γδ T cells in WT mice (n = 4 in each column) and Cd155 mRNA levels in nonhematopoietic cells in WT and Cd96−/− mice (n = 4 in each column). ( B – E ) Mice were i.p. injected with 100 µg of cIg or anti-CD96 mAb on day 1 after treatment with IMQ or petroleum jelly. (B) Changes in erythema, scaling, and thickening of dorsal skin (n = 4 in each group). (C) Representative histology (H&E staining) and epidermal thickening (n = 6 in each group) of mouse dorsal skin on day 4. (D) Il23 mRNA levels of dermal myeloid cells on day 4 (n = 4 in each group). (E) The proportion of dermal γδ T cells in CD45.2+ cells of dorsal skin (n = 4 in each group). Il17a mRNA levels of dermal γδ T cells on day 4 (n = 4 in each group) are shown. ( F and G ) WT mice were treated once a day with IMQ cream on the shaved dorsal skin from day 0 to day 4. On day 1, mice were i.p. injected with 100 µg of cIg, anti-CD96 mAb, anti–IL-17A mAb, anti–TNF-α mAb, anti-CD96 mAb plus anti–IL-17A mAb, or anti-CD96 mAb plus anti–TNF-α mAb. (F) Changes in erythema, scaling, and thickening of dorsal skin (n = 4–12 in each group). (G) Representative histology of dorsal skin (H&E staining) and epidermal thickness of dorsal skin on day 5 (n = 4–12 in each group). Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. Data are representative from two independent experiments (B) or pooled from three experiments (F and G). FIGURE 5. Blockade of CD96 ameliorates IMQ-induced psoriasis-like dermatitis. WT or Cd96−/− mice were treated once a day with IMQ or petroleum jelly on the shaved dorsal skin for 3 d. (A) Quantitative real-time PCR analysis of Cd96 mRNA levels of dermal γδ T cells in WT mice (n = 4 in each column) and Cd155 mRNA levels in nonhematopoietic cells in WT and Cd96−/− mice (n = 4 in each column). (B–E) Mice were i.p. injected with 100 µg of cIg or anti-CD96 mAb on day 1 after treatment with IMQ or petroleum jelly. (B) Changes in erythema, scaling, and thickening of dorsal skin (n = 4 in each group). (C) Representative histology (H&amp;E staining) and epidermal thickening (n = 6 in each group) of mouse dorsal skin on day 4. (D) Il23 mRNA levels of dermal myeloid cells on day 4 (n = 4 in each group). (E) The proportion of dermal γδ T cells in CD45.2+ cells of dorsal skin (n = 4 in each group). Il17a mRNA levels of dermal γδ T cells on day 4 (n = 4 in each group) are shown. (F and G) WT mice were treated once a day with IMQ cream on the shaved dorsal skin from day 0 to day 4. On day 1, mice were i.p. injected with 100 µg of cIg, anti-CD96 mAb, anti–IL-17A mAb, anti–TNF-α mAb, anti-CD96 mAb plus anti–IL-17A mAb, or anti-CD96 mAb plus anti–TNF-α mAb. (F) Changes in erythema, scaling, and thickening of dorsal skin (n = 4–12 in each group). (G) Representative histology of dorsal skin (H&amp;E staining) and epidermal thickness of dorsal skin on day 5 (n = 4–12 in each group). Error bars indicate SEM. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. ns, not significant. Data are representative from two independent experiments (B) or pooled from three experiments (F and G). More
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CD96 delivers a costimulatory signal in human γδ T cells and CD4<sup>+</sup>...
Published: 15 December 2022
FIGURE 6. CD96 delivers a costimulatory signal in human γδ T cells and CD4+ T cells. ( A and B ) Representative CD96 expression profile of human Vδ2+ γδ T cells (A) and CD4+ T cells (B). ( C ) Vδ2+ γδ T cells derived from the peripheral blood of six healthy donors (H1–H6) were stimulated with anti-CD3 mAb and either anti-CD96 mAb or cIg for 18 h and analyzed for the expression of CD69 and CD25. ( D ) Vδ2+ γδ T cells derived from eight healthy donors (H3–H5 and H7–H11) were stimulated with anti-CD3 mAb and either anti-CD96 mAb or mouse IgG1 (cIg) in the presence of TGF-β, IL-1β, IL-23, IL-6, and IL-12 for 48 h and analyzed for IL-17A production in the culture. ( E ) CD4+ T cells derived from six healthy donors (H1–H6) were stimulated with anti-CD3 mAb and either anti-CD96 mAb or cIg for 18 h and analyzed for CD69 and CD25 expressions. ( F ) CD4+ T cells derived from eight healthy donors (H2–H5 and H7–H10) were stimulated with anti-CD3 mAb and either anti-CD96 mAb or cIg in the presence of TGF-β, IL-1β, IL-23, IL-6, and IL-12 for 96 h and analyzed for IL-17A production in the culture. Error bars indicate SEM. *p < 0.05, **p < 0.01. FIGURE 6. CD96 delivers a costimulatory signal in human γδ T cells and CD4+ T cells. (A and B) Representative CD96 expression profile of human Vδ2+ γδ T cells (A) and CD4+ T cells (B). (C) Vδ2+ γδ T cells derived from the peripheral blood of six healthy donors (H1–H6) were stimulated with anti-CD3 mAb and either anti-CD96 mAb or cIg for 18 h and analyzed for the expression of CD69 and CD25. (D) Vδ2+ γδ T cells derived from eight healthy donors (H3–H5 and H7–H11) were stimulated with anti-CD3 mAb and either anti-CD96 mAb or mouse IgG1 (cIg) in the presence of TGF-β, IL-1β, IL-23, IL-6, and IL-12 for 48 h and analyzed for IL-17A production in the culture. (E) CD4+ T cells derived from six healthy donors (H1–H6) were stimulated with anti-CD3 mAb and either anti-CD96 mAb or cIg for 18 h and analyzed for CD69 and CD25 expressions. (F) CD4+ T cells derived from eight healthy donors (H2–H5 and H7–H10) were stimulated with anti-CD3 mAb and either anti-CD96 mAb or cIg in the presence of TGF-β, IL-1β, IL-23, IL-6, and IL-12 for 96 h and analyzed for IL-17A production in the culture. Error bars indicate SEM. *p &lt; 0.05, **p &lt; 0.01. More
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Proposed model. The CD96-mediated signal costimulates the TCR signal and up...
Published: 15 December 2022
FIGURE 7. Proposed model. The CD96-mediated signal costimulates the TCR signal and upregulates IL-17A production from γδ T cells, which in turn exacerbates the IMQ-induced psoriasis-like dermatitis. Consistent with this model, the pathology of IMQ-induced psoriasis-like dermatitis is suppressed wh... More
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Abundance of IL-1β, COL1A1, and COL1A2 in human amnion tissue obtained from...
Published: 15 December 2022
FIGURE 1. Abundance of IL-1β, COL1A1, and COL1A2 in human amnion tissue obtained from deliveries with spontaneous labor and membrane rupture at TL and from elective cesarean section TNL. ( A ) IL-1β abundance was significantly increased in human amnion tissue in the TL group (n = 16) compared with that in the TNL group (n = 13). ( B ) Abundance of COL1A1 and COL1A2 protein but not their mRNA was significantly increased in the TL group compared with those in the TNL group. mRNA: TL (n = 11) and TNL (n = 13); and protein: TL (n = 6) and TNL (n = 6). ( C ) Microscopy images of human amnion tissue section stained with Sirius Red showing decreased abundance of total collagen (red, bright-field) and collagen I (bright yellow, polarized light) in the TL group compared with that in the TNL group. Left panel, representative images. Right panel, average data. n = 5. *p < 0.05, **p < 0.01, TL versus TNL. FIGURE 1. Abundance of IL-1β, COL1A1, and COL1A2 in human amnion tissue obtained from deliveries with spontaneous labor and membrane rupture at TL and from elective cesarean section TNL. (A) IL-1β abundance was significantly increased in human amnion tissue in the TL group (n = 16) compared with that in the TNL group (n = 13). (B) Abundance of COL1A1 and COL1A2 protein but not their mRNA was significantly increased in the TL group compared with those in the TNL group. mRNA: TL (n = 11) and TNL (n = 13); and protein: TL (n = 6) and TNL (n = 6). (C) Microscopy images of human amnion tissue section stained with Sirius Red showing decreased abundance of total collagen (red, bright-field) and collagen I (bright yellow, polarized light) in the TL group compared with that in the TNL group. Left panel, representative images. Right panel, average data. n = 5. *p &lt; 0.05, **p &lt; 0.01, TL versus TNL. More
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Effect of IL-1β on COL1A1 and COL1A2 abundance in human amnion fibroblasts ...
Published: 15 December 2022
FIGURE 2. Effect of IL-1β on COL1A1 and COL1A2 abundance in human amnion fibroblasts and amnion tissue explants. Concentration-dependent effects of IL-1β (0, 0.1, 1, and 10 ng/ml; 24 h) on COL1A1 and COL1A2 mRNA ( A ; n = 5) and protein ( B ; n = 4) abundance in amnion fibroblasts. Time course of IL-1β effects (10 ng/ml; 0, 3, 6, 12, and 24 h) on COL1A1 and COL1A2 mRNA ( C ; n = 5) and protein ( D ; n = 5) abundance in amnion fibroblasts. ( E ) Effect of IL-1β (10 ng/ml) on COL1A1 and COL1A2 protein abundance in amnion tissue explants. Top panel is the representative Western blots. n = 3. ( F ) Microscopy image of amnion tissue explant section stained with Sirius Red showing decreased abundance of total collagen (red, bright-field) and collagen I (bright yellow, polarized light) by IL-1β (10 ng/ml) treatment. (F) Left panel, representative microscopy images. Right panel, average data. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 versus control (CTR) or 0. FIGURE 2. Effect of IL-1β on COL1A1 and COL1A2 abundance in human amnion fibroblasts and amnion tissue explants. Concentration-dependent effects of IL-1β (0, 0.1, 1, and 10 ng/ml; 24 h) on COL1A1 and COL1A2 mRNA (A; n = 5) and protein (B; n = 4) abundance in amnion fibroblasts. Time course of IL-1β effects (10 ng/ml; 0, 3, 6, 12, and 24 h) on COL1A1 and COL1A2 mRNA (C; n = 5) and protein (D; n = 5) abundance in amnion fibroblasts. (E) Effect of IL-1β (10 ng/ml) on COL1A1 and COL1A2 protein abundance in amnion tissue explants. Top panel is the representative Western blots. n = 3. (F) Microscopy image of amnion tissue explant section stained with Sirius Red showing decreased abundance of total collagen (red, bright-field) and collagen I (bright yellow, polarized light) by IL-1β (10 ng/ml) treatment. (F) Left panel, representative microscopy images. Right panel, average data. n = 3. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001 versus control (CTR) or 0. More
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Autophagy-mediated degradation of collagen I by IL-1β in human amnion fibro...
Published: 15 December 2022
FIGURE 3. Autophagy-mediated degradation of collagen I by IL-1β in human amnion fibroblasts. ( A and B ) Concentration-dependent effects of IL-1β (0, 0.1, 1, and 10 ng/ml; 24 h) on the ratio of LC3II/LC3I and ATG7 mRNA and protein. n = 4. Inhibition of IL-1β–induced (10 ng/ml; 24 h) changes in ATG7 protein ( C ; n = 5), LC3B II/I ratio (C; n = 5), and COL1A1 and COL1A2 protein ( D ; n = 5) by siRNA-mediated knockdown of RELA (p65). ( E ) Inhibition of IL-1β–induced (10 ng/ml) decreases in COL1A1 and COL1A2 protein abundance by the lysosome inhibitor CQ (50 mM). n = 4. ( F ) Inhibition of IL-1β–induced (10ng/ml) decreases in COL1A1 and COL1A2 protein abundance by siRNA-mediated knockdown of ATG7. n = 4. Top panel of each figure shows the representative Western blots. *p < 0.05, **p < 0.01, ***p < 0.001 versus 0 (A and B) versus scrambled siRNA negative control (NC) without IL-1β (C, D, and F) versus without IL-1β and CQ (E); #p < 0.05, ##p < 0.01 versus IL-1β with scrambled siRNA (C, D, and F) or IL-1β without CQ (E). FIGURE 3. Autophagy-mediated degradation of collagen I by IL-1β in human amnion fibroblasts. (A and B) Concentration-dependent effects of IL-1β (0, 0.1, 1, and 10 ng/ml; 24 h) on the ratio of LC3II/LC3I and ATG7 mRNA and protein. n = 4. Inhibition of IL-1β–induced (10 ng/ml; 24 h) changes in ATG7 protein (C; n = 5), LC3B II/I ratio (C; n = 5), and COL1A1 and COL1A2 protein (D; n = 5) by siRNA-mediated knockdown of RELA (p65). (E) Inhibition of IL-1β–induced (10 ng/ml) decreases in COL1A1 and COL1A2 protein abundance by the lysosome inhibitor CQ (50 mM). n = 4. (F) Inhibition of IL-1β–induced (10ng/ml) decreases in COL1A1 and COL1A2 protein abundance by siRNA-mediated knockdown of ATG7. n = 4. Top panel of each figure shows the representative Western blots. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001 versus 0 (A and B) versus scrambled siRNA negative control (NC) without IL-1β (C, D, and F) versus without IL-1β and CQ (E); #p &lt; 0.05, ##p &lt; 0.01 versus IL-1β with scrambled siRNA (C, D, and F) or IL-1β without CQ (E). More
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Involvement of ER-phagy in the degradation of collagen I by IL-1β in human ...
Published: 15 December 2022
FIGURE 4. Involvement of ER-phagy in the degradation of collagen I by IL-1β in human amnion fibroblasts. IL-1β (10 ng/ml) increased FAM134B but not FAM134A abundance in the scrambled siRNA negative control group (NC), but siRNA-mediated knockdown of RETREG2 (FAM134A) ( A ; n = 4) and RETREG1 (FAM134B) ( B ; n = 5) failed to block IL-1β–induced (10 ng/ml) reduction in COL1A1 and COL1A2 protein abundance. ( C ) IL-1β (10 ng/ml) increased FAM134C abundance in the scrambled siRNA group (NC), and siRNA-mediated knockdown of RETREG3 (FAM134C) blocked IL-1β–induced (10 ng/ml) reduction in COL1A1 and COL1A2 protein abundance. n = 6. Top panel of each figure shows representative Western blots. *p < 0.05, **p < 0.01, ***p < 0.001 versus scrambled siRNA (NC) without IL-1β; #p < 0.05, ##p < 0.01, ###p < 0.001 versus scrambled siRNA (NC) with IL-1β. FIGURE 4. Involvement of ER-phagy in the degradation of collagen I by IL-1β in human amnion fibroblasts. IL-1β (10 ng/ml) increased FAM134B but not FAM134A abundance in the scrambled siRNA negative control group (NC), but siRNA-mediated knockdown of RETREG2 (FAM134A) (A; n = 4) and RETREG1 (FAM134B) (B; n = 5) failed to block IL-1β–induced (10 ng/ml) reduction in COL1A1 and COL1A2 protein abundance. (C) IL-1β (10 ng/ml) increased FAM134C abundance in the scrambled siRNA group (NC), and siRNA-mediated knockdown of RETREG3 (FAM134C) blocked IL-1β–induced (10 ng/ml) reduction in COL1A1 and COL1A2 protein abundance. n = 6. Top panel of each figure shows representative Western blots. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001 versus scrambled siRNA (NC) without IL-1β; #p &lt; 0.05, ##p &lt; 0.01, ###p &lt; 0.001 versus scrambled siRNA (NC) with IL-1β. More
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Comparison of the abundance of autophagy and ER-phagy markers in human amni...
Published: 15 December 2022
FIGURE 5. Comparison of the abundance of autophagy and ER-phagy markers in human amnion tissue between deliveries with spontaneous labor and membrane rupture TL and elective cesarean section TNL. Top panel, Western blots showing the changes in ATG7, FAM134C, and LC3B II/I abundance in the amnion tissue obtained from TL (n = 6) and TNL (n = 6). Bottom panel, statistical analysis of Western blotting data. *p < 0.05, **p < 0.01 versus TNL. FIGURE 5. Comparison of the abundance of autophagy and ER-phagy markers in human amnion tissue between deliveries with spontaneous labor and membrane rupture TL and elective cesarean section TNL. Top panel, Western blots showing the changes in ATG7, FAM134C, and LC3B II/I abundance in the amnion tissue obtained from TL (n = 6) and TNL (n = 6). Bottom panel, statistical analysis of Western blotting data. *p &lt; 0.05, **p &lt; 0.01 versus TNL. More