tgfβ2 treated groups  (MedChemExpress)

Journal: Investigative Ophthalmology & Visual Science

Article Title: TXNDC5 in POAG: Promoting Extracellular Matrix Protein Accumulation and Raising Intraocular Pressure

doi: 10.1167/iovs.67.4.46

Figure Lengend Snippet: Proteomic analysis showed that expressions of TXNDC5 and ECM proteins were upregulated in TGFβ2-treated HTM cells. HTM cells were starved with serum-free DMEM/F12 medium for 24 hours and then treated with TGFβ2 (5 ng/mL) for 48 hours. (A) Changes of protein expression after TGFβ2 treatment in HTM cells were represented by volcano plots (≥1.2-fold difference: upregulated genes [ red ] and downregulated genes [ blue ]). Violin plots showed the TGFβ2-induced significant changes in TXNDC5 (B) , LN (C) , FN (D) , COL4A1 (E) , and COL4A2 (F) . ** P < 0.01; *** P < 0.001. (G) KEGG analysis indicated multiple metabolic and signal pathways, including ECM-receptor interaction, were changed markedly.

Article Snippet: Both control and TGFβ2-treated groups were induced with 100 mg/mL CHX (MedChemExpress, Monmouth Junction, NJ, USA) to inhibit protein translation.

Techniques: Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: TXNDC5 in POAG: Promoting Extracellular Matrix Protein Accumulation and Raising Intraocular Pressure

doi: 10.1167/iovs.67.4.46

Figure Lengend Snippet: Western blot, qRT-PCR, and immunocytochemistry showed that expressions of TXNDC5 and ECM proteins were upregulated in TGFβ2-treated HTM cells. (A) Concentration of TXNDC5 in the non-POAG ( n = 10) and POAG patients ( n = 7) in aqueous humor detected by ELISA. (B, C) TXNDC5 levels were not correlated with age and IOP in the non-POAG group and in POAG patients ( n = 7–8). (D) Representative images of Western blot analysis showing TGFβ2-induced changes in expressions of TXNDC5 and ECM proteins in HTM cells. (E–H) Quantitative analyses of Western blot of individual protein ( n = 4–6): TXNDC5 ( E ) , LN (F) , FN (G) , Col-IV (H) . Data are presented as mean ± SEM. (A) * P < 0.05 by unpaired, two-tailed t -test compared to non-POAG. (E–H) * P < 0.05; ** P < 0.01; *** P < 0.001 by unpaired, two-tailed t -test compared to control. (I) Representative images of immunofluorescence staining of TXNDC5, LN, FN, and COL-IV proteins in untreated control and TGFβ2-treated groups. Scale bars : 50 µm.

Article Snippet: Both control and TGFβ2-treated groups were induced with 100 mg/mL CHX (MedChemExpress, Monmouth Junction, NJ, USA) to inhibit protein translation.

Techniques: Western Blot, Quantitative RT-PCR, Immunocytochemistry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Control, Immunofluorescence, Staining

Journal: PLOS One

Article Title: Single-cell profiling unveils key regulators of skeletal stem cells in chicken and human embryonic limb development

doi: 10.1371/journal.pone.0346514

Figure Lengend Snippet: Expression of COL5A2, COL1A2, PRRX1, and TGFβ2 was detected in the periosteal region and the primary ossification centers. A. Merged and single-channel images of COL5A2 (red) and DAPI (blue) staining. B. Merged and single-channel images of COL1A2 (red) and DAPI (blue) staining. C. Merged and single-channel images of PRRX1 (red) and DAPI (blue) staining. D. Merged and single-channel images of TGFβ2 (red) and DAPI (blue) staining. All experiments were independently repeated at least three times. Scale bars in snapshot image, 200 μm; scale bars in magnified images, 50 μm.

Article Snippet: The following antibodies were used: anti-COL5A2 antibody ( GB111012 , 1:500, Servicebio, Wuhan, China), anti-COL1A2 antibody (AF7001, 1:200, Affinity Biosciences, Jiangsu, China), anti-PRRX1 antibody (DF4274, 1:200, Affinity Biosciences, Jiangsu, China), anti-TGFβ2 antibody (AF0260, 1:100, Affinity Biosciences, Jiangsu, China), and Cy3-conjugated goat anti-rabbit IgG (GB21303, 1:300, Servicebio, Wuhan, China).

Techniques: Expressing, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: TXNDC5 in POAG: Promoting Extracellular Matrix Protein Accumulation and Raising Intraocular Pressure

doi: 10.1167/iovs.67.4.46

Figure Lengend Snippet: Proteomic analysis showed that expressions of TXNDC5 and ECM proteins were upregulated in TGFβ2-treated HTM cells. HTM cells were starved with serum-free DMEM/F12 medium for 24 hours and then treated with TGFβ2 (5 ng/mL) for 48 hours. (A) Changes of protein expression after TGFβ2 treatment in HTM cells were represented by volcano plots (≥1.2-fold difference: upregulated genes [ red ] and downregulated genes [ blue ]). Violin plots showed the TGFβ2-induced significant changes in TXNDC5 (B) , LN (C) , FN (D) , COL4A1 (E) , and COL4A2 (F) . ** P < 0.01; *** P < 0.001. (G) KEGG analysis indicated multiple metabolic and signal pathways, including ECM-receptor interaction, were changed markedly.

Article Snippet: HTM cells were starved in serum-free medium, treated with TGFβ2 (5 ng/mL; Med Chem Express, Monmouth Junction, NJ, USA) for 48 hours, and then subjected to subsequent assays.

Techniques: Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: TXNDC5 in POAG: Promoting Extracellular Matrix Protein Accumulation and Raising Intraocular Pressure

doi: 10.1167/iovs.67.4.46

Figure Lengend Snippet: Western blot, qRT-PCR, and immunocytochemistry showed that expressions of TXNDC5 and ECM proteins were upregulated in TGFβ2-treated HTM cells. (A) Concentration of TXNDC5 in the non-POAG ( n = 10) and POAG patients ( n = 7) in aqueous humor detected by ELISA. (B, C) TXNDC5 levels were not correlated with age and IOP in the non-POAG group and in POAG patients ( n = 7–8). (D) Representative images of Western blot analysis showing TGFβ2-induced changes in expressions of TXNDC5 and ECM proteins in HTM cells. (E–H) Quantitative analyses of Western blot of individual protein ( n = 4–6): TXNDC5 ( E ) , LN (F) , FN (G) , Col-IV (H) . Data are presented as mean ± SEM. (A) * P < 0.05 by unpaired, two-tailed t -test compared to non-POAG. (E–H) * P < 0.05; ** P < 0.01; *** P < 0.001 by unpaired, two-tailed t -test compared to control. (I) Representative images of immunofluorescence staining of TXNDC5, LN, FN, and COL-IV proteins in untreated control and TGFβ2-treated groups. Scale bars : 50 µm.

Article Snippet: HTM cells were starved in serum-free medium, treated with TGFβ2 (5 ng/mL; Med Chem Express, Monmouth Junction, NJ, USA) for 48 hours, and then subjected to subsequent assays.

Techniques: Western Blot, Quantitative RT-PCR, Immunocytochemistry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Control, Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: TXNDC5 in POAG: Promoting Extracellular Matrix Protein Accumulation and Raising Intraocular Pressure

doi: 10.1167/iovs.67.4.46

Figure Lengend Snippet: Knockdown or overexpression of TXNDC5 regulated TGFβ2-induced ECM aggregation in HTM cells. HTM cells were pre-transfected with si-TXNDC5 for eight hours and then treated with or without TGFβ2 for 48 hours. (A) Western blot assessment of the knockdown efficiency of three different tested siRNA sequences of si-TXNDC5. The most effective of them (no. 3) was used for the following knockdown studies. (B–G) Western blot and immunocytochemical staining analyses for TXNDC5 and ECM proteins by si-TXNDC5 transfection with or without TGFβ2 induction. (H–J) Western blot was assayed for ECM (FN) expression by overexpressing TXNDC5. Data are presented as mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001 compared with control; # P < 0.05; ## P < 0.01; ### P < 0.001 compared with TGFβ2 group by one-way ANOVA followed by Tukey's multiple comparisons test. Scale bar : 50 µm.

Article Snippet: HTM cells were starved in serum-free medium, treated with TGFβ2 (5 ng/mL; Med Chem Express, Monmouth Junction, NJ, USA) for 48 hours, and then subjected to subsequent assays.

Techniques: Knockdown, Over Expression, Transfection, Western Blot, Staining, Expressing, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: TXNDC5 in POAG: Promoting Extracellular Matrix Protein Accumulation and Raising Intraocular Pressure

doi: 10.1167/iovs.67.4.46

Figure Lengend Snippet: TXNDC5 knockdown reduced the TGFβ2-induced TGFβR2 expression, whereas TGFβR2 knockdown did not affect TXNDC5 expression. HTM cells were pre-transfected with si-TXNDC5 or si-TGFβR2 for eight hours with or without TGFβ2 treatment for 48 hours. (A) Representative images showing protein levels of TXNDC5, TGFβR1 and TGFβR2 after si-TXNDC5 with or without TGFβ2 treatment in the HTM cells. (B, C) Quantitative analyses of TGFβR1 and TGFβR2 levels after si-TXNDC5 with or without TGFβ2 treatment. (D) Three different siRNA sequences were tested for their knockdown efficiency of TGFβR2. The most effective of them (no. 3) was used for the following knockdown studies. (E–H) The effect of TGFβR2 knockdown on the expression of TGFβR2, TXNDC5 and FN proteins with or without TGFβR2 treatment. Error bars : mean ± SEM ( n = 3-6); * P < 0.05; ** P < 0.01; *** P < 0.001 compared with control; # P < 0.05; ### P < 0.001 compare with TGFβ2. One-way ANOVA followed by Tukey's multiple comparisons test. Scale bar : 50 µm.

Article Snippet: HTM cells were starved in serum-free medium, treated with TGFβ2 (5 ng/mL; Med Chem Express, Monmouth Junction, NJ, USA) for 48 hours, and then subjected to subsequent assays.

Techniques: Knockdown, Expressing, Transfection, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: TXNDC5 in POAG: Promoting Extracellular Matrix Protein Accumulation and Raising Intraocular Pressure

doi: 10.1167/iovs.67.4.46

Figure Lengend Snippet: TXNDC5 is degraded via the lysosomal pathway. (A–D) Quantitative analyses qRT-PCR of individual mRNA ( n = 3–6): TXNDC5 (A) , LN (B) , FN (C) , COL-IV (D) . (E, F) After TGFβ2 treatment for 24 hours, CHX (100 µg/mL) was added to the HTM cells to inhibit the synthesis of new proteins. Cells were harvested at the indicated time points (0, 12, or 24 hours after CHX treatment) and immunoblotted to evaluate TXNDC5 protein levels. (E) Representative images of the immunoblot. (F) The slope of decline of TXNDC5 protein in TGFβ2 and Control groups. (G) Quantitative analysis, the level of TXNDC5 relative to GAPDH at time 0 of each group defines 1. (H, I) Effects of 24 hours treatment with lysosomal inhibitor CQ (10 µM) or proteasome inhibitor MG132 (5 µM) on TXNDC5 accumulation in HTM cells. The level of TXNDC5 relative to GAPDH of the control group (DMSO only) defines 1. (J, K) Effects of CQ and TGFβ2 on TXNDC5 accumulation in HTM cells. (L) Appropriate amount of cell lysate was taken for input group, TXNDC5 was immunoprecipitated with Hsc70, and TXNDC5 and Hsc70 protein expression was detected by Western blot. The level of TXNDC5 relative to GAPDH of the control group defines 1. All quantitative data are shown as mean and SEM. (A–F) * P < 0.05; ** P < 0.01; *** P < 0.001 by unpaired, two-tailed t -test compared to control. (G–J) * P < 0.05; ** P < 0.01; *** P < 0.001 vs. Control; # P < 0.05 between the indicated groups by one-way ANOVA followed with Tukey's multiple comparisons test.

Article Snippet: HTM cells were starved in serum-free medium, treated with TGFβ2 (5 ng/mL; Med Chem Express, Monmouth Junction, NJ, USA) for 48 hours, and then subjected to subsequent assays.

Techniques: Quantitative RT-PCR, Western Blot, Control, Immunoprecipitation, Expressing, Two Tailed Test

Journal: Investigative Ophthalmology & Visual Science

Article Title: TXNDC5 in POAG: Promoting Extracellular Matrix Protein Accumulation and Raising Intraocular Pressure

doi: 10.1167/iovs.67.4.46

Figure Lengend Snippet: Involvement of CMA influences TXNDC5 protein levels induced by TGFβ2. (A) The amino acid sequence of TXNDC5 has a "KFERQ" -like pentapeptide motif. (B) Three different siRNA sequences of LAMP2A were used to test their knockdown efficiency of LAMP2A, the most effective of which (No. 2) was used for following knockdown studies. (C, D) Silencing of LAMP2A aggravated upregulation of TXNDC5 by TGFβ2 induction. (E, F) Treatment with AR7 (40 µM) at HTM for 24 hours reduced accumulation of TXNDC5 by siLAMP2A. (G, H) AR7 treatment for 24 hours reversed TGFβ2-induced upregulation of TXNDC5. All quantitative data are shown as mean and SEM ( n = 3–6). * P < 0.05; ** P < 0.01; *** P < 0.001 vs. Control; # P < 0.05; ## P < 0.01; ### P < 0.001 between the indicated groups by one-way ANOVA followed with Tukey's multiple comparisons test.

Article Snippet: HTM cells were starved in serum-free medium, treated with TGFβ2 (5 ng/mL; Med Chem Express, Monmouth Junction, NJ, USA) for 48 hours, and then subjected to subsequent assays.

Techniques: Sequencing, Knockdown, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: TXNDC5 in POAG: Promoting Extracellular Matrix Protein Accumulation and Raising Intraocular Pressure

doi: 10.1167/iovs.67.4.46

Figure Lengend Snippet: Knockdown of TXNDC5 attenuated ECM protein accumulation and high IOP induced by Ad.hTGFβ2 226/228 in mouse TM. (A) Illustration of experimental design: si-TXNDC5 or si-negative control (si-NC) was injected intravitreally on day 0 and day 7, and Ad.hTGFβ2 226/228 was injected intravitreally once on day 2. Mice were euthanized on day 14. (B) Changes in IOP treated by Ad.hTGFβ2 226/228 with or without si-TXNDC5 treatment. (C) Statistical analysis of mouse IOP on day 10. (D–G) Expression of TXNDC5 (D) , FN (E) , COL-IV (F) , LN (G) mRNA in TM tissues from the study groups. (H) Representative immunofluorescence stainings of TXNDC5, LN, FN, and COL-IV in mouse ocular tissue are shown. Blue fluorescence = DAPI; red fluorescence = TXNDC5 or indicated ECM protein. Data are represented as means ± SEM ( n ≥ 4 mice for each group). * P < 0.05; ** P < 0.01; *** P < 0.001 compared with control; # P < 0.05; ## P < 0.01; ### P < 0.001 compared with TGFβ2 group; & P < 0.05; && P < 0.01 compared with TGFβ2 + si-TXNDC5 group. One-way ANOVA followed by Tukey's multiple comparisons test. Scale bar : 50 µm (H) .

Article Snippet: HTM cells were starved in serum-free medium, treated with TGFβ2 (5 ng/mL; Med Chem Express, Monmouth Junction, NJ, USA) for 48 hours, and then subjected to subsequent assays.

Techniques: Knockdown, Negative Control, Injection, Expressing, Immunofluorescence, Fluorescence, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: TXNDC5 in POAG: Promoting Extracellular Matrix Protein Accumulation and Raising Intraocular Pressure

doi: 10.1167/iovs.67.4.46

Figure Lengend Snippet: Schematic representation of the involvement of CMA lysosomal degradation pathway of TXNDC5 in TM ECM protein accumulation. Treatment with TGFβ2 increased the expression of TXNDC5 and further promoted the expression of TGFβR2, which in turn promoted the increase of ECM protein. Intracellularlly, mutual recognition of the molecular chaperone Hsc70 with TXNDC5 protein and synergism with LAMP2A molecule promotes the entry of TXNDC5 protein into lysosomal degradation, and activation of CMA activity by AR7 leads to the entry of more TXNDC5 into lysosomal degradation, thereby reducing ECM accumulation. The expression of TXNDC5 in the aqueous humor of POAG patients was significantly higher than that of non-POAG patients. In the Ad.hTGFβ 226/228 -mediated high IOP mouse model, injection of si-TXNDC5 significantly improved Ad.hTGFβ 226/228 -mediated high IOP and ECM accumulation in TM tissue.

Article Snippet: HTM cells were starved in serum-free medium, treated with TGFβ2 (5 ng/mL; Med Chem Express, Monmouth Junction, NJ, USA) for 48 hours, and then subjected to subsequent assays.

Techniques: Expressing, Activation Assay, Activity Assay, Injection