Journal: Heliyon

Article Title: Green synthesis of silver nanoparticle using Oscillatoria sp. extract, its antibacterial, antibiofilm potential and cytotoxicity activity

doi: 10.1016/j.heliyon.2019.e02502

Figure Lengend Snippet: The antibacterial activity of OsSNPs against E . coli ATCC 35218.

Article Snippet: The antibacterial activity of silver nanoparticles from this research showed that Escherichia coli ATCC 25318 was more susceptible to the silver nanoparticles while Bacillus sp. was least susceptible to the biosynthesized silver nanoparticles.

Techniques: Activity Assay

Journal: Developmental cell

Article Title: Mechanical pressure-induced dedifferentiation of myofibroblasts inhibits scarring via SMYD3/ITGBL1 signaling.

doi: 10.1016/j.devcel.2023.04.014

Figure Lengend Snippet: Figure 2. PT inhibits SMYD3 expression in scar-derived myofibroblasts and hypertrophic scar tissues (A) Flow chart of RNA-seq experimental scheme. (B) RNA-seq analysis of scar-derived myofibroblasts with or without PT. (C) qRT-PCR validation of the RNA sequencing results (n = 5). (D) Representative immunofluorescence staining of SMYD3 in normal skin fibroblasts and scar-derived myofibroblasts with or without PT (n = 5). The nucleus is indicated by a dashed circle. Scale bars: 30 mm. (E) Western blot analysis for SMYD3 expression (n = 3). (F) Representative immunohistochemical staining for SMYD3 in patient biopsy samples. Scale bars: 100 mm. (G) Quantification of the expression of SMYD3 in (F) (n = 6). (H) Western blot analysis for SMYD3 expression in patient biopsy samples (n = 6). Data are shown as mean ± SEM. *p < 0.05, by two-tailed unpaired Student’s t test (C), and by one-way ANOVA followed by the Tukey-Kramer’s post hoc test (D, E, G, and H).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER DMSO Sigma Cat# D2650 CAS: 67-68-5 ChIP Grade Protein G Magnetic Beads Cell Signaling Technology Cat# 9006S RIPA lysis and extraction buffer Thermo Fisher Scientific Cat# 89900 Triton X-100 Sigma Cat# T8787 CAS: CA9036-19-5 Critical commercial assays KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 FragELTM DNA Fragmentation Detection Kit EMD Millipore Cat# QIA39 Bicinchoninic acid protein assay kit Sigma Cat# BCA1 Annexin V–FITC Apoptosis Detection Kit BD Biosciences Cat# 556547 RNeasy Mini Kit Qiagen Cat# 74104 First-Strand cDNA Synthesis Kit Fermentas Cat# K1622 SYBR Green RT–PCR Reagent Kit Toyobo Cat# QPK-201 Luciferase assay kit GeneCopoeia Cat# LF013 Pierce Agarose ChIP Kit Thermo Fisher Scientific Cat# 26156 ON-TARGETplus Human SMYD3 siRNA – SMARTpool Dharmacon Cat# L-013737-00-0020 ON-TARGETplus non-targeting pool Dharmacon Cat# D-001810-10-20 DharmaFECT 1 Transfection Reagent Dharmacon Cat# T-2001-04 GeneEditorTM in vitro Site-Directed Mutagenesis System Promega Cat# Q9280 Deposited data RNA-seq data This paper NCBI Sequence Read Archive: PRJNA803352 Original images for immunohistochemistry staining, immunofluorescence staining and western blot This paper Mendeley: https://doi.org/10.17632/ 3vckxmyh42.2 Experimental models: Organisms/strains New Zealand White female rabbits Experimental Animal Research Laboratory of Sun Yat-sen University N/A Oligonucleotides shRNAs specific for SMYD3 3’ UTR region (5’-GCTGTGTGAACCTCTCTTATT-3’) This paper N/A Primers for qRT-PCR analysis, see Table S1 This paper N/A Primers for ChIP-PCR analysis, see Tables S2–S6 This paper N/A Bacterial and virus strains SMYD3-pcDNA3.1(+) plasmid (NM_001167740) This paper N/A ITGBL1-pcDNA3.1(+) plasmid (NM_002211.4) This paper N/A ILK-pcDNA3.1(+) plasmid (NM_001014794) This paper N/A TCF4-pcDNA3.1(+) plasmid (NM_001243226) This paper N/A pcDNA3.1 vector Thermo Fisher Scientific Cat# V79020 pGL3-Basic plasmid Promega Cat# E1751 SMYD3 pro-Luc plasmid This paper N/A Lipofectamine 2000 Life Technology Cat# 11668-019 pLKO.1 puro vector Addgene Cat# 8453 (Continued on next page) e2 Developmental Cell 58, 1139–1152.e1–e6, July 10, 2023

Techniques: Expressing, Derivative Assay, RNA Sequencing, Quantitative RT-PCR, Biomarker Discovery, Staining, Western Blot, Immunohistochemical staining, Two Tailed Test

Journal: Developmental cell

Article Title: Mechanical pressure-induced dedifferentiation of myofibroblasts inhibits scarring via SMYD3/ITGBL1 signaling.

doi: 10.1016/j.devcel.2023.04.014

Figure Lengend Snippet: Figure 3. SMYD3 overexpression inhibits PT-induced dedifferentiation of scar-derived myofibroblasts into normal fibroblasts (A) Overexpression of SMYD3 in scar-derived myofibroblasts was confirmed by western blot analysis (n = 3). (B) Effects of SMYD3 overexpression on the expression of myofibroblast biomarkers in PT-treated scar-derived myofibroblasts. Scale bars: 100 mm. (C–F) Quantification of the expression of myofibroblast biomarkers in (B) (n = 5). (G) Analysis of cell migration using a wound healing assay. Scale bars: 200 mm. (H) Quantification of the effects of SMYD3 overexpression on the migratory ability of PT-treated scar-derived myofibroblasts (n = 5). (I) Western blot analysis for collagen I and III proteins. (J and K) Quantification of collagen I and III western blot analysis (n = 3). (L) Effects of SMYD3 overexpression on collagen synthesis of PT-treated scar-derived myofibroblasts (n = 5). Data are shown as mean ± SEM. *p < 0.05, by two-tailed unpaired Student’s t test (A), and by one-way ANOVA followed by the Tukey-Kramer’s post hoc test (C–F, H, J, and K).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER DMSO Sigma Cat# D2650 CAS: 67-68-5 ChIP Grade Protein G Magnetic Beads Cell Signaling Technology Cat# 9006S RIPA lysis and extraction buffer Thermo Fisher Scientific Cat# 89900 Triton X-100 Sigma Cat# T8787 CAS: CA9036-19-5 Critical commercial assays KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 FragELTM DNA Fragmentation Detection Kit EMD Millipore Cat# QIA39 Bicinchoninic acid protein assay kit Sigma Cat# BCA1 Annexin V–FITC Apoptosis Detection Kit BD Biosciences Cat# 556547 RNeasy Mini Kit Qiagen Cat# 74104 First-Strand cDNA Synthesis Kit Fermentas Cat# K1622 SYBR Green RT–PCR Reagent Kit Toyobo Cat# QPK-201 Luciferase assay kit GeneCopoeia Cat# LF013 Pierce Agarose ChIP Kit Thermo Fisher Scientific Cat# 26156 ON-TARGETplus Human SMYD3 siRNA – SMARTpool Dharmacon Cat# L-013737-00-0020 ON-TARGETplus non-targeting pool Dharmacon Cat# D-001810-10-20 DharmaFECT 1 Transfection Reagent Dharmacon Cat# T-2001-04 GeneEditorTM in vitro Site-Directed Mutagenesis System Promega Cat# Q9280 Deposited data RNA-seq data This paper NCBI Sequence Read Archive: PRJNA803352 Original images for immunohistochemistry staining, immunofluorescence staining and western blot This paper Mendeley: https://doi.org/10.17632/ 3vckxmyh42.2 Experimental models: Organisms/strains New Zealand White female rabbits Experimental Animal Research Laboratory of Sun Yat-sen University N/A Oligonucleotides shRNAs specific for SMYD3 3’ UTR region (5’-GCTGTGTGAACCTCTCTTATT-3’) This paper N/A Primers for qRT-PCR analysis, see Table S1 This paper N/A Primers for ChIP-PCR analysis, see Tables S2–S6 This paper N/A Bacterial and virus strains SMYD3-pcDNA3.1(+) plasmid (NM_001167740) This paper N/A ITGBL1-pcDNA3.1(+) plasmid (NM_002211.4) This paper N/A ILK-pcDNA3.1(+) plasmid (NM_001014794) This paper N/A TCF4-pcDNA3.1(+) plasmid (NM_001243226) This paper N/A pcDNA3.1 vector Thermo Fisher Scientific Cat# V79020 pGL3-Basic plasmid Promega Cat# E1751 SMYD3 pro-Luc plasmid This paper N/A Lipofectamine 2000 Life Technology Cat# 11668-019 pLKO.1 puro vector Addgene Cat# 8453 (Continued on next page) e2 Developmental Cell 58, 1139–1152.e1–e6, July 10, 2023

Techniques: Over Expression, Derivative Assay, Western Blot, Expressing, Migration, Wound Healing Assay, Two Tailed Test

Journal: Developmental cell

Article Title: Mechanical pressure-induced dedifferentiation of myofibroblasts inhibits scarring via SMYD3/ITGBL1 signaling.

doi: 10.1016/j.devcel.2023.04.014

Figure Lengend Snippet: Figure 4. SMYD3 promotes scar-derived myofibroblast differentiation by increasing ITGBL1 expression (A) RNA sequencing analysis of PT-treated scar-derived myofibroblasts with or without SMYD3 overexpression. (B) qRT-PCR validation of the RNA sequencing results (n = 5). (C) Representative immunofluorescence staining of ITGBL1 in normal skin fibroblasts and scar-derived myofibroblasts with or without PT (n = 5). Scale bars: 30 mm. (D) Western blot analysis for ITGBL1 expression in above cells (n = 3). (E) Representative immunohistochemical staining for ITGBL1 in the normal skin fibroblasts and PT-treated scar-derived myofibroblasts with or without SMYD3 overexpression (n = 5). Scale bars: 30 mm. (F) Western blot analysis for ITGBL1 expression in above cells (n = 3). (G) Representative immunohistochemical staining for ITGBL1 in patient biopsy samples (n = 6). Scale bars: 100 mm. (H) Western blot analysis for ITGBL1 expression in patient biopsy samples (n = 6). (I) Overexpression of ITGBL1 was confirmed by western blot analysis (n = 3). (J) Effects of ITGBL1 overexpression on the expression of myofibroblast biomarkers in PT-treated scar-derived myofibroblasts. Scale bars: 100 mm. (K–N) Quantification of the expression of myofibroblast biomarkers in (J) (n = 5). (O) Analysis of cell migration using a wound healing assay. Scale bars: 200 mm. (P) Quantification of the effects of ITGBL1 overexpression on the migratory ability of PT-treated scar-derived myofibroblasts (n = 5). (Q) Western blot analysis for collagen I and III proteins. (R and S) Quantification of collagen I and III western blot analysis in (Q) (n = 3). (T) Effects of ITGBL1 overexpression on collagen synthesis of PT-treated scar-derived myofibroblasts (n = 5). (U) Western blot analysis for SMYD3 protein (n = 3). Data are shown as mean ± SEM. *p < 0.05, by two-tailed unpaired Student’s t test (B, E, F, I, and U), and by one-way ANOVA followed by the Tukey-Kramer’s post hoc test (C, D, G, H, K–N, P, and R–T).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER DMSO Sigma Cat# D2650 CAS: 67-68-5 ChIP Grade Protein G Magnetic Beads Cell Signaling Technology Cat# 9006S RIPA lysis and extraction buffer Thermo Fisher Scientific Cat# 89900 Triton X-100 Sigma Cat# T8787 CAS: CA9036-19-5 Critical commercial assays KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 FragELTM DNA Fragmentation Detection Kit EMD Millipore Cat# QIA39 Bicinchoninic acid protein assay kit Sigma Cat# BCA1 Annexin V–FITC Apoptosis Detection Kit BD Biosciences Cat# 556547 RNeasy Mini Kit Qiagen Cat# 74104 First-Strand cDNA Synthesis Kit Fermentas Cat# K1622 SYBR Green RT–PCR Reagent Kit Toyobo Cat# QPK-201 Luciferase assay kit GeneCopoeia Cat# LF013 Pierce Agarose ChIP Kit Thermo Fisher Scientific Cat# 26156 ON-TARGETplus Human SMYD3 siRNA – SMARTpool Dharmacon Cat# L-013737-00-0020 ON-TARGETplus non-targeting pool Dharmacon Cat# D-001810-10-20 DharmaFECT 1 Transfection Reagent Dharmacon Cat# T-2001-04 GeneEditorTM in vitro Site-Directed Mutagenesis System Promega Cat# Q9280 Deposited data RNA-seq data This paper NCBI Sequence Read Archive: PRJNA803352 Original images for immunohistochemistry staining, immunofluorescence staining and western blot This paper Mendeley: https://doi.org/10.17632/ 3vckxmyh42.2 Experimental models: Organisms/strains New Zealand White female rabbits Experimental Animal Research Laboratory of Sun Yat-sen University N/A Oligonucleotides shRNAs specific for SMYD3 3’ UTR region (5’-GCTGTGTGAACCTCTCTTATT-3’) This paper N/A Primers for qRT-PCR analysis, see Table S1 This paper N/A Primers for ChIP-PCR analysis, see Tables S2–S6 This paper N/A Bacterial and virus strains SMYD3-pcDNA3.1(+) plasmid (NM_001167740) This paper N/A ITGBL1-pcDNA3.1(+) plasmid (NM_002211.4) This paper N/A ILK-pcDNA3.1(+) plasmid (NM_001014794) This paper N/A TCF4-pcDNA3.1(+) plasmid (NM_001243226) This paper N/A pcDNA3.1 vector Thermo Fisher Scientific Cat# V79020 pGL3-Basic plasmid Promega Cat# E1751 SMYD3 pro-Luc plasmid This paper N/A Lipofectamine 2000 Life Technology Cat# 11668-019 pLKO.1 puro vector Addgene Cat# 8453 (Continued on next page) e2 Developmental Cell 58, 1139–1152.e1–e6, July 10, 2023

Techniques: Derivative Assay, Expressing, RNA Sequencing, Over Expression, Quantitative RT-PCR, Biomarker Discovery, Staining, Western Blot, Immunohistochemical staining, Migration, Wound Healing Assay, Two Tailed Test

Journal: Developmental cell

Article Title: Mechanical pressure-induced dedifferentiation of myofibroblasts inhibits scarring via SMYD3/ITGBL1 signaling.

doi: 10.1016/j.devcel.2023.04.014

Figure Lengend Snippet: Figure 5. SMYD3 regulates ITGBL1 expression through direct regulation of H3K4me3 levels (A–C) ChIP-PCR assay of H3K4 methylation levels of ITGBL1 in normal skin fibroblasts and scar-derived myofibroblasts (n = 4). (D) Western blot analysis for H3K4me2/3 levels. (E) The agarose gel electrophoresis assay for ChIP-PCR products, showing the specific occupancy of SMYD3 on ITGBL1 promoters #3 and #4 (995 to 456 bp) in normal fibroblasts and scar-derived myofibroblasts. M, marker. (F and G) qRT-PCR of the ChIP products validated the binding capacity of SMYD3 to the ITGLB1 promoter (n = 4). (H and I) Effects of SMYD3 overexpression on ITGBL1 H3K4me2/3 levels in PT-treated scar-derived myofibroblasts (n = 4). (J) Western blot analysis for H3K4me2/3 levels. (K) ChIP-PCR assays of SMYD3 binding to the ITGBL1 promoter (n = 4). (L and M) Effects of SMYD3 knockdown on ITGBL1 H3K4me2/3 levels in PT-treated scar-derived myofibroblasts (n = 4). (N) Western blot analysis for H3K4me2/3 levels. (O) qRT-PCR of the ChIP products validated the binding of SMYD3 to the ITGLB1 promoter (n = 4). (P) Representative immunohistochemical staining for ITGBL1 expression. Scale bars: 50 mm. (Q) Western blot analysis for ITGBL1 proteins (n = 3). Data are shown as mean ± SEM. *p < 0.05, by two-tailed unpaired Student’s t test (F–K), and by one-way ANOVA followed by the Tukey-Kramer’s post hoc test (A–C and L–Q).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER DMSO Sigma Cat# D2650 CAS: 67-68-5 ChIP Grade Protein G Magnetic Beads Cell Signaling Technology Cat# 9006S RIPA lysis and extraction buffer Thermo Fisher Scientific Cat# 89900 Triton X-100 Sigma Cat# T8787 CAS: CA9036-19-5 Critical commercial assays KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 FragELTM DNA Fragmentation Detection Kit EMD Millipore Cat# QIA39 Bicinchoninic acid protein assay kit Sigma Cat# BCA1 Annexin V–FITC Apoptosis Detection Kit BD Biosciences Cat# 556547 RNeasy Mini Kit Qiagen Cat# 74104 First-Strand cDNA Synthesis Kit Fermentas Cat# K1622 SYBR Green RT–PCR Reagent Kit Toyobo Cat# QPK-201 Luciferase assay kit GeneCopoeia Cat# LF013 Pierce Agarose ChIP Kit Thermo Fisher Scientific Cat# 26156 ON-TARGETplus Human SMYD3 siRNA – SMARTpool Dharmacon Cat# L-013737-00-0020 ON-TARGETplus non-targeting pool Dharmacon Cat# D-001810-10-20 DharmaFECT 1 Transfection Reagent Dharmacon Cat# T-2001-04 GeneEditorTM in vitro Site-Directed Mutagenesis System Promega Cat# Q9280 Deposited data RNA-seq data This paper NCBI Sequence Read Archive: PRJNA803352 Original images for immunohistochemistry staining, immunofluorescence staining and western blot This paper Mendeley: https://doi.org/10.17632/ 3vckxmyh42.2 Experimental models: Organisms/strains New Zealand White female rabbits Experimental Animal Research Laboratory of Sun Yat-sen University N/A Oligonucleotides shRNAs specific for SMYD3 3’ UTR region (5’-GCTGTGTGAACCTCTCTTATT-3’) This paper N/A Primers for qRT-PCR analysis, see Table S1 This paper N/A Primers for ChIP-PCR analysis, see Tables S2–S6 This paper N/A Bacterial and virus strains SMYD3-pcDNA3.1(+) plasmid (NM_001167740) This paper N/A ITGBL1-pcDNA3.1(+) plasmid (NM_002211.4) This paper N/A ILK-pcDNA3.1(+) plasmid (NM_001014794) This paper N/A TCF4-pcDNA3.1(+) plasmid (NM_001243226) This paper N/A pcDNA3.1 vector Thermo Fisher Scientific Cat# V79020 pGL3-Basic plasmid Promega Cat# E1751 SMYD3 pro-Luc plasmid This paper N/A Lipofectamine 2000 Life Technology Cat# 11668-019 pLKO.1 puro vector Addgene Cat# 8453 (Continued on next page) e2 Developmental Cell 58, 1139–1152.e1–e6, July 10, 2023

Techniques: Expressing, Methylation, Derivative Assay, Western Blot, Agarose Gel Electrophoresis, Marker, Quantitative RT-PCR, Binding Assay, Over Expression, Knockdown, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Developmental cell

Article Title: Mechanical pressure-induced dedifferentiation of myofibroblasts inhibits scarring via SMYD3/ITGBL1 signaling.

doi: 10.1016/j.devcel.2023.04.014

Figure Lengend Snippet: Figure 6. PT inhibits SMYD3 expression by regulating ILK-mediated TCF4 expression (A) The agarose gel electrophoresis assay for ChIP-PCR products, showing the specific occupancy of TCF4 on SMYD3 promoters #2 and #3 (1,840 to 1,734 bp) in normal fibroblasts and scar-derived myofibroblasts. M, marker. (B) qRT-PCR of the ChIP products validated the binding capacity of TCF4 on SMYD3 promoters (n = 4). (C) Luciferase reporter assay for SMYD3 promoter activity (n = 4). (D) Overexpression of ILK was confirmed by western blot analysis (n = 3). (E–G) Western blot analysis for p-AKT and p-GSK-3b expression (n = 3). (H and I) Western blot analysis for TCF-4 expression (n = 3). (J) qRT-PCR of the ChIP products validated the binding capacity of TCF4 on SMYD3 promoters (n = 4). (K) Effects of ILK overexpression on SMYD3 promoter activity (n = 4). (L and M) Western blot analysis for SMYD3 protein expression (n = 3). Data are shown as mean ± SEM. *p < 0.05, by two-tailed unpaired Student’s t test (D, J, and K), and by one-way ANOVA followed by the Tukey-Kramer’s post hoc test (B, C, F, G, I, and M).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER DMSO Sigma Cat# D2650 CAS: 67-68-5 ChIP Grade Protein G Magnetic Beads Cell Signaling Technology Cat# 9006S RIPA lysis and extraction buffer Thermo Fisher Scientific Cat# 89900 Triton X-100 Sigma Cat# T8787 CAS: CA9036-19-5 Critical commercial assays KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 FragELTM DNA Fragmentation Detection Kit EMD Millipore Cat# QIA39 Bicinchoninic acid protein assay kit Sigma Cat# BCA1 Annexin V–FITC Apoptosis Detection Kit BD Biosciences Cat# 556547 RNeasy Mini Kit Qiagen Cat# 74104 First-Strand cDNA Synthesis Kit Fermentas Cat# K1622 SYBR Green RT–PCR Reagent Kit Toyobo Cat# QPK-201 Luciferase assay kit GeneCopoeia Cat# LF013 Pierce Agarose ChIP Kit Thermo Fisher Scientific Cat# 26156 ON-TARGETplus Human SMYD3 siRNA – SMARTpool Dharmacon Cat# L-013737-00-0020 ON-TARGETplus non-targeting pool Dharmacon Cat# D-001810-10-20 DharmaFECT 1 Transfection Reagent Dharmacon Cat# T-2001-04 GeneEditorTM in vitro Site-Directed Mutagenesis System Promega Cat# Q9280 Deposited data RNA-seq data This paper NCBI Sequence Read Archive: PRJNA803352 Original images for immunohistochemistry staining, immunofluorescence staining and western blot This paper Mendeley: https://doi.org/10.17632/ 3vckxmyh42.2 Experimental models: Organisms/strains New Zealand White female rabbits Experimental Animal Research Laboratory of Sun Yat-sen University N/A Oligonucleotides shRNAs specific for SMYD3 3’ UTR region (5’-GCTGTGTGAACCTCTCTTATT-3’) This paper N/A Primers for qRT-PCR analysis, see Table S1 This paper N/A Primers for ChIP-PCR analysis, see Tables S2–S6 This paper N/A Bacterial and virus strains SMYD3-pcDNA3.1(+) plasmid (NM_001167740) This paper N/A ITGBL1-pcDNA3.1(+) plasmid (NM_002211.4) This paper N/A ILK-pcDNA3.1(+) plasmid (NM_001014794) This paper N/A TCF4-pcDNA3.1(+) plasmid (NM_001243226) This paper N/A pcDNA3.1 vector Thermo Fisher Scientific Cat# V79020 pGL3-Basic plasmid Promega Cat# E1751 SMYD3 pro-Luc plasmid This paper N/A Lipofectamine 2000 Life Technology Cat# 11668-019 pLKO.1 puro vector Addgene Cat# 8453 (Continued on next page) e2 Developmental Cell 58, 1139–1152.e1–e6, July 10, 2023

Techniques: Expressing, Agarose Gel Electrophoresis, Derivative Assay, Marker, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Over Expression, Western Blot, Two Tailed Test

Journal: Developmental cell

Article Title: Mechanical pressure-induced dedifferentiation of myofibroblasts inhibits scarring via SMYD3/ITGBL1 signaling.

doi: 10.1016/j.devcel.2023.04.014

Figure Lengend Snippet: Figure 7. Scar hypertrophy is inhibited by pressure therapy or SMYD3 knockdown (A) Flow chart of in vivo experimental scheme. (B) Clinical appearance of the wounds at day 0 and scar formation at day 28. Scale bars: 5 mm. (C) Representative histological staining of the scar tissues on postoperative day 28. Scale bars: 200 mm (H&E, upper) and 100 mm (Masson’s trichrome, lower). (D) Quantification of epidermal thickness based on the histological staining (C) (n = 6). (E) H&E staining of the hair follicles (blue arrows) and sebaceous glands (yellow arrows). Scale bars: 200 mm (103, left) and 50 mm (403, right). (F) Quantification of hair follicles numbers in (E) (n = 6). (G) The schematic diagram of SEI, which is calculated as the ratio of area of newly formed dermis (total scar area)/estimated area of non-scarred dermis (un- wounded dermis). (H) The analysis of SEI on postoperative day 28 (n = 6). (I) The scar elasticity analysis using the Cutometer MPA 580 (n = 6). (J) Western blot analysis for collagen I and III proteins (n = 6). (K) Western blot analysis for SMYD3 and ITGBL1 proteins (n = 6). (L) Clinical appearance of the scar formation after the inhibition of SMYD3. Scale bars: 5 mm. (M) Representative histological staining of the scar tissues on postoperative day 28. Scale bars: 200 mm (H&E, upper) and 100 mm (Masson’s trichrome, lower). (N) Quantification of epidermal thickness based on the histological staining (M) (n = 6). (O) H&E staining of the hair follicles (blue arrows) and sebaceous glands (yellow arrows). Scale bars: 200 mm (left) and 50 mm (right). (P) Quantification of hair follicles numbers in (O) (n = 6). (Q) The analysis of the SEI on postoperative day 28 (n = 6). (R) The scar elasticity analysis using the Cutometer MPA 580 (n = 6). (S) Western blot analysis for collagen I and III proteins (n = 6). (T) Western blot analysis for SMYD3 and ITGBL1 proteins (n = 6). Data are shown as mean ± SEM. *p < 0.05, by two-tailed unpaired Student’s t test (D, F, and H), and by one-way ANOVA followed by the Tukey-Kramer’s post hoc test (I–K, N, and P–T).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER DMSO Sigma Cat# D2650 CAS: 67-68-5 ChIP Grade Protein G Magnetic Beads Cell Signaling Technology Cat# 9006S RIPA lysis and extraction buffer Thermo Fisher Scientific Cat# 89900 Triton X-100 Sigma Cat# T8787 CAS: CA9036-19-5 Critical commercial assays KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 FragELTM DNA Fragmentation Detection Kit EMD Millipore Cat# QIA39 Bicinchoninic acid protein assay kit Sigma Cat# BCA1 Annexin V–FITC Apoptosis Detection Kit BD Biosciences Cat# 556547 RNeasy Mini Kit Qiagen Cat# 74104 First-Strand cDNA Synthesis Kit Fermentas Cat# K1622 SYBR Green RT–PCR Reagent Kit Toyobo Cat# QPK-201 Luciferase assay kit GeneCopoeia Cat# LF013 Pierce Agarose ChIP Kit Thermo Fisher Scientific Cat# 26156 ON-TARGETplus Human SMYD3 siRNA – SMARTpool Dharmacon Cat# L-013737-00-0020 ON-TARGETplus non-targeting pool Dharmacon Cat# D-001810-10-20 DharmaFECT 1 Transfection Reagent Dharmacon Cat# T-2001-04 GeneEditorTM in vitro Site-Directed Mutagenesis System Promega Cat# Q9280 Deposited data RNA-seq data This paper NCBI Sequence Read Archive: PRJNA803352 Original images for immunohistochemistry staining, immunofluorescence staining and western blot This paper Mendeley: https://doi.org/10.17632/ 3vckxmyh42.2 Experimental models: Organisms/strains New Zealand White female rabbits Experimental Animal Research Laboratory of Sun Yat-sen University N/A Oligonucleotides shRNAs specific for SMYD3 3’ UTR region (5’-GCTGTGTGAACCTCTCTTATT-3’) This paper N/A Primers for qRT-PCR analysis, see Table S1 This paper N/A Primers for ChIP-PCR analysis, see Tables S2–S6 This paper N/A Bacterial and virus strains SMYD3-pcDNA3.1(+) plasmid (NM_001167740) This paper N/A ITGBL1-pcDNA3.1(+) plasmid (NM_002211.4) This paper N/A ILK-pcDNA3.1(+) plasmid (NM_001014794) This paper N/A TCF4-pcDNA3.1(+) plasmid (NM_001243226) This paper N/A pcDNA3.1 vector Thermo Fisher Scientific Cat# V79020 pGL3-Basic plasmid Promega Cat# E1751 SMYD3 pro-Luc plasmid This paper N/A Lipofectamine 2000 Life Technology Cat# 11668-019 pLKO.1 puro vector Addgene Cat# 8453 (Continued on next page) e2 Developmental Cell 58, 1139–1152.e1–e6, July 10, 2023

Techniques: Knockdown, In Vivo, Staining, Western Blot, Inhibition, Two Tailed Test