Journal: Biomedicines

Article Title: SMYD2 Promotes Calcium Oxalate-Induced Glycolysis in Renal Tubular Epithelial Cells via PTEN Methylation

doi: 10.3390/biomedicines12102279

Figure Lengend Snippet: SMYD2 and glycolysis are activated in human nephrolithiasis samples. Representative images of ( A ) HE staining, ( B ) immunofluorescence for CD68, and ( C ) Masson’s staining in the KS group. ( D ) Representative immunofluorescence images and semi-quantitative analysis of SMYD2 expression. ( E ) Renal lipid metabolism was identified using Oil Red O staining, and the area of positive staining on renal sections was quantified. The black arrows point to the presence of lipid deposits in damaged RTCs. ( F – H ) Representative immunohistochemistry images and semi-quantitative analysis of PPARα, CPT1, HK2, LDHA, and PKM2. Scale bar = 100 μm. Data are expressed as mean ± SD, ** p < 0.01; *** p < 0.001.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies against SMYD2 (Proteintech), HK2 (ABclonal), PKM2 (Proteintech), LDHA (ABclonal), FN (Boster), α-SMA (Proteintech), COL1A1 (Affinity Biologicals, Ancaster, Canada), BAX (ABclonal), BCL-2 (ABclonal), Cleaved caspase3 (Abcam), mTOR (Proteintech), p-mTOR (Proteintech), S6 (ABclonal), p-S6 (ABclonal), AKT (Proteintech), p-AKT (Proteintech), PTEN (ABclonal), N-cadherin (ABclonal), Vimentin (Proteintech), Vinculin (ABclonal), and β-Actin (Servicebio).

Techniques: Staining, Immunofluorescence, Expressing, Immunohistochemistry

Journal: Biomedicines

Article Title: SMYD2 Promotes Calcium Oxalate-Induced Glycolysis in Renal Tubular Epithelial Cells via PTEN Methylation

doi: 10.3390/biomedicines12102279

Figure Lengend Snippet: SMYD2 and glycolysis are activated in glyoxylate-induced nephrolithiasis mice. ( A ) Representative immunofluorescence images and semi-quantitative analysis of SMYD2 expression. ( B ) Representative images of Oil Red O staining and quantification of the positive staining area in renal tissues of CON and Gly groups; black arrows indicate lipid deposition in RTCs. ( C , D ) Representative images and semi-quantitative immunohistochemistry analysis of HK2, LDHA, PKM2, CPT1, and PPARα in CON and Gly groups. ( E , F ) Western blot examines HK2, PKM2, and LDHA levels in renal tissues, with quantification by densitometry. Scale bar = 50 μm. Data are expressed as mean ± SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies against SMYD2 (Proteintech), HK2 (ABclonal), PKM2 (Proteintech), LDHA (ABclonal), FN (Boster), α-SMA (Proteintech), COL1A1 (Affinity Biologicals, Ancaster, Canada), BAX (ABclonal), BCL-2 (ABclonal), Cleaved caspase3 (Abcam), mTOR (Proteintech), p-mTOR (Proteintech), S6 (ABclonal), p-S6 (ABclonal), AKT (Proteintech), p-AKT (Proteintech), PTEN (ABclonal), N-cadherin (ABclonal), Vimentin (Proteintech), Vinculin (ABclonal), and β-Actin (Servicebio).

Techniques: Immunofluorescence, Expressing, Staining, Immunohistochemistry, Western Blot

Journal: Biomedicines

Article Title: SMYD2 Promotes Calcium Oxalate-Induced Glycolysis in Renal Tubular Epithelial Cells via PTEN Methylation

doi: 10.3390/biomedicines12102279

Figure Lengend Snippet: Inhibition of SMYD2 with AZ505 reduces glycolysis in glyoxylate-induced nephrolithiasis mice. ( A – D ) Immunofluorescence and Western blot analyses to quantify the expression of SMYD2 in renal tissues of the indicated groups. ( E , F ) Immunohistochemistry and semi-quantitative analysis of the levels of HK2, LDHA, and PKM2 in renal tissues of the indicated groups. ( G , H ) Western blot analysis and densitometric analysis of the HK2, PKM2, and LDHA levels in renal tissues. Scale bar = 50 μm. Data are expressed as mean ± SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies against SMYD2 (Proteintech), HK2 (ABclonal), PKM2 (Proteintech), LDHA (ABclonal), FN (Boster), α-SMA (Proteintech), COL1A1 (Affinity Biologicals, Ancaster, Canada), BAX (ABclonal), BCL-2 (ABclonal), Cleaved caspase3 (Abcam), mTOR (Proteintech), p-mTOR (Proteintech), S6 (ABclonal), p-S6 (ABclonal), AKT (Proteintech), p-AKT (Proteintech), PTEN (ABclonal), N-cadherin (ABclonal), Vimentin (Proteintech), Vinculin (ABclonal), and β-Actin (Servicebio).

Techniques: Inhibition, Immunofluorescence, Western Blot, Expressing, Immunohistochemistry

Journal: Biomedicines

Article Title: SMYD2 Promotes Calcium Oxalate-Induced Glycolysis in Renal Tubular Epithelial Cells via PTEN Methylation

doi: 10.3390/biomedicines12102279

Figure Lengend Snippet: Silencing SMYD2 specifically inhibits glycolysis, apoptosis, inflammation, and EMT in HK-2 cells. ( A , B ) Western blot verified the knockdown efficiency of SMYD2 and quantified it by densitometry. ( C – F ) Western blot showed that SMYD2-knockdown suppressed HK2, PKM2, and LDHA expression. ( G ) Measurement of lactate in the supernatant of HK-2 cells from the specified groups ( H ) Representative immunofluorescence images of HK2, PKM2, LDHA after SMYD2-knockdown. ( I , J ) Western blot detection of N-cadherin, Vimentin, FN, Collagen l, α-SMA, BAX, BCL-2, Cleaved caspase3 expression after SMYD2 knockdown and quantification by densitometry. ( K ) The levels of IL-1β, IL-6, and TNF-α in the supernatant of HK-2 cells in the indicated group were measured by ELISA. Scale bar = 50 μm. Data are expressed as mean ± SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies against SMYD2 (Proteintech), HK2 (ABclonal), PKM2 (Proteintech), LDHA (ABclonal), FN (Boster), α-SMA (Proteintech), COL1A1 (Affinity Biologicals, Ancaster, Canada), BAX (ABclonal), BCL-2 (ABclonal), Cleaved caspase3 (Abcam), mTOR (Proteintech), p-mTOR (Proteintech), S6 (ABclonal), p-S6 (ABclonal), AKT (Proteintech), p-AKT (Proteintech), PTEN (ABclonal), N-cadherin (ABclonal), Vimentin (Proteintech), Vinculin (ABclonal), and β-Actin (Servicebio).

Techniques: Western Blot, Knockdown, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: SMYD2 Promotes Calcium Oxalate-Induced Glycolysis in Renal Tubular Epithelial Cells via PTEN Methylation

doi: 10.3390/biomedicines12102279

Figure Lengend Snippet: SMYD2 methylates PTEN and inhibits its expression, thereby promoting glycolysis by activating the mTORC1 pathway in HK-2 cells. ( A – D ) Western blot and densitometry analysis detected the expression of PTEN and mTORC1 signals after si-SMYD2 in vitro and in vivo. ( E ) Demonstration of two protein binding models in Surface form. ( F ) SMYD2 interaction with PTEN in HK-2 cells. ( G ) In HK-2 cells transfected with si-SMYD2 or si-Ctrl, IP with anti-PTEN antibody, and subsequent blotting with anti-PTEN, anti-methylated lysine antibodies. ( H ) Western blot analysis of the levels of HK2, PKM2, and LDHA in HK-2 cells. Data are expressed as mean ± SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies against SMYD2 (Proteintech), HK2 (ABclonal), PKM2 (Proteintech), LDHA (ABclonal), FN (Boster), α-SMA (Proteintech), COL1A1 (Affinity Biologicals, Ancaster, Canada), BAX (ABclonal), BCL-2 (ABclonal), Cleaved caspase3 (Abcam), mTOR (Proteintech), p-mTOR (Proteintech), S6 (ABclonal), p-S6 (ABclonal), AKT (Proteintech), p-AKT (Proteintech), PTEN (ABclonal), N-cadherin (ABclonal), Vimentin (Proteintech), Vinculin (ABclonal), and β-Actin (Servicebio).

Techniques: Expressing, Western Blot, In Vitro, In Vivo, Protein Binding, Transfection, Methylation

Journal: Biomedicines

Article Title: SMYD2 Promotes Calcium Oxalate-Induced Glycolysis in Renal Tubular Epithelial Cells via PTEN Methylation

doi: 10.3390/biomedicines12102279

Figure Lengend Snippet: In CaOx-induced kidney injury, PTEN is methylated by SMYD2, leading to its inhibition and subsequent activation of the mTORC1 pathway. This inhibition prompts a metabolic shift toward glycolysis in RTCs, ultimately culminating in renal stone formation, kidney injury, and kidney fibrosis. Therefore, targeting the SMYD2-mediated epigenetic dysregulation of metabolism may offer a promising approach to treating renal stone disease. Inhibitors of SMYD2 and glycolysis could inhibit renal stone formation and decrease renal injury and fibrosis.

Article Snippet: Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies against SMYD2 (Proteintech), HK2 (ABclonal), PKM2 (Proteintech), LDHA (ABclonal), FN (Boster), α-SMA (Proteintech), COL1A1 (Affinity Biologicals, Ancaster, Canada), BAX (ABclonal), BCL-2 (ABclonal), Cleaved caspase3 (Abcam), mTOR (Proteintech), p-mTOR (Proteintech), S6 (ABclonal), p-S6 (ABclonal), AKT (Proteintech), p-AKT (Proteintech), PTEN (ABclonal), N-cadherin (ABclonal), Vimentin (Proteintech), Vinculin (ABclonal), and β-Actin (Servicebio).

Techniques: Methylation, Inhibition, Activation Assay

Journal: JCI Insight

Article Title: Inhibition of Wnt/β-catenin signaling ameliorates osteoarthritis in a murine model of experimental osteoarthritis

doi: 10.1172/jci.insight.96308

Figure Lengend Snippet: (A) Synovial fibroblasts isolated from patients with osteoarthritis (OA) were positive for expression of type I collagen (red) but not type II collagen (green). Nuclei were stained with DAPI (blue). Isotype controls were negative. Original magnification, ×20. (B) OA synovial fibroblasts were compared with control synovial fibroblasts by real-time qRT-PCR of AXIN2, a downstream marker of Wnt signaling. Expression was higher in synovial fibroblasts isolated from patients with OA than those from controls. Statistical analysis was performed using a t test, ***P < 0.001, n = 6. (C) Schematic of the Wnt/β-catenin pathway showing target molecules inhibited by the Wnt inhibitors (C113 and XAV-939) used in the study. (D) Furthermore, synovial fibroblasts were treated with recombinant WNT3A with and without Wnt inhibitors (C113 and XAV-939) and analyzed by real-time RT-PCR of AXIN2. Wnt signaling was ameliorated by both inhibitors in control and OA synovial fibroblasts. One-way ANOVA with Tukey’s post-hoc test was used to compare treatment groups, ***P < 0.001, n = 6. (E) Immunoblotting of β-catenin in OA synovial fibroblast cell lysates after treatment with recombinant WNT3A with or without inhibitors. Densitometry of immunoblots was performed to quantify the reduction of Wnt signaling after inhibitor treatment. One-way ANOVA with Tukey’s post-hoc test was used to compare treatment groups, *P < 0.05, **P < 0.01, n = 8. Alteration of β-catenin signaling in the nucleus was confirmed by immunoblotting of synovial fibroblast nuclear lysates compared with lamin B1 loading control. (B, D, and E) For data presented as box-and-whiskers plots, horizontal lines indicate the medians, cross marks indicate the means, boxes indicate the 25th to 75th percentiles, and whiskers indicate the minimum and maximum values of the data set.

Article Snippet: Tissue sections were blocked with 10% goat serum; incubated with antibodies against COL1A1 (MDBioproducts, 203002), COL2A1 (Millipore, MAB8887), Vimentin (ThermoFisher Scientific, PA1-10003), active Jnk (Promega, V7931), Periostin (Santa Cruz, sc-67233), β-catenin (BD Transduction Laboratories, 610153), PCNA (Santa Cruz, sc-56), or SOX9 (Millipore, AB5535); incubated with secondary antibody (goat anti-mouse FITC (Leinco Technologies, M359) or goat anti-rabbit Cy3 (Jackson ImmunoResearch 111-165-144); and mounted with ProLong Gold antifade reagent with DAPI (Invitrogen).

Techniques: Isolation, Expressing, Staining, Quantitative RT-PCR, Marker, Recombinant, Western Blot

Journal: JCI Insight

Article Title: Inhibition of Wnt/β-catenin signaling ameliorates osteoarthritis in a murine model of experimental osteoarthritis

doi: 10.1172/jci.insight.96308

Figure Lengend Snippet: (A) Real-time qRT-PCR of COL1A1 on synovial fibroblasts isolated from osteoarthritis (OA) patients shows an increase after recombinant WNT3A treatment and a decrease after Wnt inhibitor treatment. One-way ANOVA with Tukey’s post-hoc test was performed, *P < 0.05, n = 5. (B) Immunoblotting of type I collagen in OA synovial fibroblasts also demonstrates an increase after activation of Wnt signaling and a decrease in response to C113 or XAV-939 inhibitors. Densitometry was used to quantify COL1A1 protein levels normalized to the β-actin loading control. One-way ANOVA with Tukey’s post-hoc test was performed, *P < 0.05, **P < 0.01, n = 8. For data presented as box-and-whiskers plots, horizontal lines indicate the medians, cross marks indicate the means, boxes indicate the 25th to 75th percentiles, and whiskers indicate the minimum and maximum values of the data set.

Article Snippet: Tissue sections were blocked with 10% goat serum; incubated with antibodies against COL1A1 (MDBioproducts, 203002), COL2A1 (Millipore, MAB8887), Vimentin (ThermoFisher Scientific, PA1-10003), active Jnk (Promega, V7931), Periostin (Santa Cruz, sc-67233), β-catenin (BD Transduction Laboratories, 610153), PCNA (Santa Cruz, sc-56), or SOX9 (Millipore, AB5535); incubated with secondary antibody (goat anti-mouse FITC (Leinco Technologies, M359) or goat anti-rabbit Cy3 (Jackson ImmunoResearch 111-165-144); and mounted with ProLong Gold antifade reagent with DAPI (Invitrogen).

Techniques: Quantitative RT-PCR, Isolation, Recombinant, Western Blot, Activation Assay