Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Photoreceptor-specific knockout of Dync1h1 . ( A ) Schematic of mouse cytoplasmic dynein heavy chain (DYNC1H1, gene symbol Dync1h1 ). Locations of the truncation point ( red arrow ), six AAA motor domains ( blue ), and microtubule-binding domain ( green ) are indicated. Locations of previously identified mutations in mice and zebrafish are shown below the protein. Dync1h1 knockout results in truncation of DYNC1H1 in its tail domain and eliminates the motor domain. ( B ) Cytoplasmic dynein drawn schematically with heavy chain ( blue ). Adapted from Hoang et al. ( C ) loxP sites of the floxed Dync1h1 allele are located in introns 23 and 25. ( D ) Following Cre-induced recombination, the null allele loses exons 24 and 25. ( E ) PCR amplification using P10 retinal DNA as template and primers X23F and loxP-R. The amplicon at 500 bp shows Cre-induced recombination has occurred in the Dync1h1 F/F ;iCre75 retina, while amplicon at 1600 bp represents Dync1h1 F/F present in the inner retina. ( F ) Genotyping of Dync1h1 F/F and Dync1h1 F/+ alleles with primers loxP-F and loxP-R in intron 25. M, size markers; W, water control. ( G ) Genotyping of iCre75. +, tail DNA containing the iCre75 transgene. ( H ) Genotyping of Egfp-Cetn2. +, tail DNA containing the Egfp-Cetn2 transgene. ( I ) Genotyping of Prom1-ETCre mice.

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Knock-Out, Binding Assay, Amplification

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Scotopic and photopic electroretinography. ( A – C ) Average scotopic a-wave amplitudes as a function of light intensity (−4.5 to 2.4 log cd s·m −2 ) at P16 (A), P21 (B), and P30 (C). The scotopic rod Dync1h1 − / − a-wave amplitude decreases by P21 and is nearly extinguished at P30. ( D ) Summary of peak amplitudes at 2.4 log cd s·m −2 ; average peak scotopic a-wave amplitude and standard deviations are indicated. ( E , F ) Average photopic b-wave amplitudes as a function of light intensity (−0.1 to 1.9 log cd s·m −2 ) at P21 (E) and P30 (F). Decline of the P30 photopic b-wave is probably caused by bystander cone degeneration following loss of rods in the rod knockout.

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Knock-Out

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Immunohistochemistry with DYNC1H1 and rhodopsin (Rho) antibodies. ( A–D ) Representative control (rows A, C) and rod Dync1h1 −/− retinas (rows B, D) labeled with antibodies directed against DYNC1H1 (rows A, B) and rhodopsin (rows C, D) at P14, P16, P18, P21, and P30. DYNC1H1 is located in the photoreceptor IS and OPL (photoreceptor terminals and bipolar cell dendrites). The rod Dync1h1 − / − ONL starts to shrink at P16, and only cone nuclei remain at P30. Rhodopsin-containing rod OSs are severely shortened by P21 and absent by P30. Note, almost no rhodopsin accumulates in the ONL. Scale bar : 20 µm. ( E ) Comparison of the average ONL thickness of control and rod Dync1h1 −/− central retinas illustrates loss of rod photoreceptors between P16 and P30. Error bars show the standard deviation. * P < 0.05 (Student's t -test). ( F ) Comparison of the average combined length of rod OS + IS for P10 to P30 control and rod Dync1h1 −/− retinas shows loss of OS and IS in the absence of DYNC1H1. Error bars show the standard deviation. * P < 0.05 (Student's t -test). For E and F, the numbers of mice for control and rodKO, respectively, were P10, n = 5, 4; P12, n = 8, 3; P14, n = 5, 3; P16, n = 8, 3; P18, n = 3, 3; P21, n = 7, 3; P30, n = 10, 3.

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Immunohistochemistry, Labeling, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Immunohistochemistry with PDE6 and RIBEYE antibodies. ( A–D ) Representative control (rows A, C) and rod Dync1h1 −/− retinas (rows B, D) labeled with antibodies directed against PDE6 (MOE; rows A, B) and RIBEYE (rows C, D). Rod and cone OS are shortened by P16 and lost by P30. RIBEYE expression in the ribbon synapses is lost between P16 and P30 with diminished expression evident by P21.

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Immunohistochemistry, Labeling, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Tamoxifen-induced DYNC1H1 depletion in the adult retina. ( A–D ) tam Dync1h1 −/− (tamoxifen-induced KO, first column ), uninjected Dync1h1 F/F ;Prom1-CreER T2 ( second column ), tamoxifen-injected Dync1h1 F/F retinas ( third column ), and tamoxifen-injected Dync1h1 F/+ ;Prom1-CreER T2 ( fourth column ) harvested at 1wPTI (A), 2wPTI (B), 3wPTI (C), or 4wPTI (D). Cryosections were labeled with anti-DYNC1H1 ( red ) and contrasted with DAPI ( blue ). Uninjected Dync1h1 F/F ;Prom1-CreER T2 and injected Dync1h1 F/F or Dync1h1 F/+ ;Prom1-CreER T2 retinas all maintain normal IS and ONL thickness throughout the experiment. In tam Dync1h1 −/− (induced KO) retina, the ONL and IS shrank between 2wPTI and 3wPTI, but this shrinking was not statistically significant until 4wPTI. In retinas with efficient knockout of Dync1h1, ONL thinning increased in severity by 4wPTI. ( E ) Plot of average central ONL thickness at 1, 2, 3, and 4wPTI showing ONL thickness decreases in the tam Dync1h1 −/− from 2wPTI to 4wPTI. * P < 0.05 (Welch's ANOVA). ( F ) Plot of average combined OS + IS lengths shows that tam Dync1h1 −/− rod and cone OS shrink between 2wPTI and 4wPTI. * P < 0.05 (Welch's ANOVA). For E and F, the numbers of mice for control injected, control uninjected, and tam Dync1h1 −/− , respectively, were 1wPTI, n = 5, 5, 5; 2wPTI, n = 3, 3, 3; 3wPTI, n = 5, 5, 4; 4wPTI, n = 11, 5, 6.

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Injection, Labeling, Knock-Out

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Effect of tamoxifen-induced DYNC1H1 deletion on rhodopsin expression. ( A–D ) Representative immunohistochemistry from tam Dync1h1 −/− ( first column ), uninjected Dync1h1 F/F ;Prom1-CreER T2 ( second column ), tamoxifen-injected Dync1h1 F/F retinas ( third column ), and tamoxifen-injected Dync1h1 F/+ ;Prom1-CreER T2 ( fourth column ) harvested at 1wPTI (A), 2wPTI (B), 3wPTI (C), or 4wPTI (D) and probed with anti-rhodopsin ( red ). Nuclei were contrasted with DAPI ( blue ). tam Dync1h1 −/− retina reveals rod OS shortening and ONL thinning from 2 to 3wPTI. Rod OS shortening and ONL thinning increased in severity by 4wPTI, with minor rhodopsin expression in the ONL. ( E – G ) Average scotopic ERG traces at 1.4 log cd s·m −2 from control and tam Dync1h1 −/− at 2wPTI (E), 3wPTI (F), and 4wPTI (G). Scotopic responses decrease between 2 and 4 wPTI in the tam Dync1h1 −/− .

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Expressing, Immunohistochemistry, Injection

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Effect of tamoxifen-induced DYNC1H1 deletion on cone-opsin expression. ( A–D ) Representative immunohistochemistry from tam Dync1h1 −/− ( first column ), uninjected Dync1h1 F/F ;Prom1-CreER T2 ( second column ), tamoxifen-injected Dync1h1 F/F retinas ( third column ), and tamoxifen-injected Dync1h1 F/+ ;Prom1-CreER T2 ( fourth column ) harvested at 1wPTI (A), 2wPTI (B), 3wPTI (C), or 4wPTI (D) probed with anti–M-opsin ( red ). ONL indicated by DAPI ( blue ). tam Dync1h1 −/− retina reveals loss/shortening of cone OS from 2 to 3wPTI. While some M-opsin is seen in the ONL at 4wPTI, cone OSs are absent. ( E–G ) Average photopic ERG traces at 1.4 log cd s·m −2 from control and tam Dync1h1 −/− at 2wPTI (E), 3wPTI (F), and 4wPTI (G). Photopic responses decrease between 2wPTI and 4wPTI in the tam Dync1h1 −/− .

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Expressing, Immunohistochemistry, Injection

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Summary of ERG responses as a function of intensity in tam Dync1h1 −/− retina. ( A–D ) Average scotopic a-wave amplitudes ( left column ) and photopic b-wave amplitudes ( right column ) as a function of light intensity shrink between 2wPTI and 4wPTI. Injected control ( blue ), uninjected control ( green ), and tamoxifen-induced knockout ( red ). At 1wPTI (A), average scotopic and photopic ERGs amplitudes did not differ significantly between induced knockout and control. At 2wPTI (B), the average scotopic a-wave amplitude was slightly reduced compared to controls. Photopic ERGs did not differ. At 3wPTI (C), induced KO shows reduced average ERG amplitudes for both scotopic a-wave and photopic b-wave, but this reduction was only statically significant for the photopic 1.4 log cd s·m −2 intensity. At 4wPTI (D), the reduction in average ERG amplitude for both scotopic a-wave and photopic b-wave for the induced KO was statically significant for most ERG flash intensities. * P < 0.05 (unbalanced two-factor ANOVA, Tukey's honestly significant difference criterion).

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Injection, Knock-Out

Journal: Theranostics

Article Title: Elevated IgE promotes cardiac fibrosis by suppressing miR-486a-5p.

doi: 10.7150/thno.47845

Figure Lengend Snippet: Figure 1. Effects of IgE-FcεR1 signaling on CFs. (A) FcεR1a expression levels in CFs of WT and FcεR1-KO mice measured by immunoblot. (B-D) FcεR1a expression levels after IgE treatment (5 μg/mL) for 0, 3, and 24 h measured by qPCR (B) and immunoblot (C-D). (E-F) mRNA expression of key fibrotic genes (a-SMA, Col1a1 and Col3a1) in IgE-stimulated FcεR1-WT (E) and FcεR1-KO (F) CFs at 0, 3, and 24 h. (G) Representative western blot of α-SMA, COL1A1, and COL3A1 in FcεR1-WT CFs after IgE treatment for 0, 3, and 24 h. (H) Statistical analysis of α-SMA, COL1A1, and COL3A1 protein expression in FcεR1-WT CFs, normalized to GAPDH (fold change versus 0 h). (I) Representative western blot of α-SMA, COL1A1, and COL3A1 in FcεR1-KO CFs after IgE treatment. (J) Statistical analysis of the relative protein expression of α-SMA, COL1A1, and COL3A1 in FcεR1-KO CFs. Data are mean ± SD from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s indicates no significant difference in One-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Antibodies, sources, and dilutions were as follows: rabbit anti-FcεR1 antibody (Cat#10980-1-AP, Proteintech, 1:1000), rabbit anti-αSMA (Cat#ab5694, Abcam, 1:1000), rabbit antiSMAD1 (Cat#6944S, Cell Signaling, 1:1000), rabbit anti-phospho-SMAD1 (Cat#PA5-36771, Invitrogen, 1:1000), rabbit anti-SMAD2 (Cat#12570-1-AP, Proteintech, 1:1000), rabbit anti-phospho-SMAD2 (Cat#18338S, Cell Signaling, 1:1000), rabbit anti-TGFβ1 (Cat#ab179695, Abcam, 1:1000), rabbit antiCollagen I (Cat#PA1-26204, Invitrogen, 1:1000), rabbit anti-Collagen III (Cat#22734-1-AP, Proteintech, 1:1000), rabbit anti-GFP (Cat#ab290, Abcam, 1:1000), and rabbit anti-GAPDH (Cat#10494-1-AP, Proteintech, 1:3000).

Techniques: Expressing, Western Blot

Journal: Theranostics

Article Title: Elevated IgE promotes cardiac fibrosis by suppressing miR-486a-5p.

doi: 10.7150/thno.47845

Figure Lengend Snippet: Figure 2. Effects of CF FcεR1 deletion on Ang II-induced cardiac fibrosis. (A) Representative images of Masson staining of the heart tissues from Ang II- or saline-infused FcεR1-Flox and FcεR1-cKO mice. Images were taken at 200X magnification. Scale bars, 50 μm. (B) Quantification of myocardial interstitial fibrosis by Masson staining. A total of nine fields from three sections (three fields from each section) per mouse were randomly selected for analysis. (C) Representative images of Sirius Red staining of the heart tissues from Ang II- or saline-infused FcεR1-Flox and FcεR1-cKO mice. Images were taken at 400X magnification. Scale bars, 50 μm. (D) Quantification of myocardial interstitial fibrosis by Sirius Red staining. A total of nine fields from three sections (three fields from each section) per mouse were randomly selected for analysis. (E) qPCR analysis of periostin (Postn) and a-SMA expression in the heart tissues of the four indicated groups. (F) Representative immunoblot analysis showing the expression of COL1A1 and COL3A1 in the hearts of Ang II- or saline-infused FcεR1-Flox and FcεR1-cKO mice. (G) Statistical analysis of COL1A1 and COL3A1 expression normalized to GAPDH. Total n = 5 (Saline/FcεR1-Flox), n = 5 (Saline/cKO), n = 10 (Ang II/FcεR1-Flox), or n = 9 (Ang II/cKO) per group. Results are shown as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001 by Two-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Antibodies, sources, and dilutions were as follows: rabbit anti-FcεR1 antibody (Cat#10980-1-AP, Proteintech, 1:1000), rabbit anti-αSMA (Cat#ab5694, Abcam, 1:1000), rabbit antiSMAD1 (Cat#6944S, Cell Signaling, 1:1000), rabbit anti-phospho-SMAD1 (Cat#PA5-36771, Invitrogen, 1:1000), rabbit anti-SMAD2 (Cat#12570-1-AP, Proteintech, 1:1000), rabbit anti-phospho-SMAD2 (Cat#18338S, Cell Signaling, 1:1000), rabbit anti-TGFβ1 (Cat#ab179695, Abcam, 1:1000), rabbit antiCollagen I (Cat#PA1-26204, Invitrogen, 1:1000), rabbit anti-Collagen III (Cat#22734-1-AP, Proteintech, 1:1000), rabbit anti-GFP (Cat#ab290, Abcam, 1:1000), and rabbit anti-GAPDH (Cat#10494-1-AP, Proteintech, 1:3000).

Techniques: Staining, Saline, Expressing, Western Blot

Journal: Theranostics

Article Title: Elevated IgE promotes cardiac fibrosis by suppressing miR-486a-5p.

doi: 10.7150/thno.47845

Figure Lengend Snippet: Figure 3. IgE alters miRNAs profile in CFs and down-regulates miR-486a-5p. (A) Heatmap illustrates differentially expressed miRNAs. The expression is normalized as row z-scores, up-regulation is indicated in red and down-regulation is indicated in blue. Each row represents a miRNA. (B) Three-way Venn diagram indicating the total number of miRNAs predicted to directly target Col1a1 and Col3a1. The numbers of shared miRNAs are indicated in the intersections of the Venn diagram. Two miRNAs in the intersections are shown as black stars in (A). (C) Two-way Venn diagram indicating the miRNAs that may be involved in fibrosis. Seven miRNAs in the intersection are shown as red stars in (A). (D) qPCR analysis of eight candidate miRNAs from the intersections shown in (B-C). The expression levels of miRNAs in FcεR1-WT (left panel) and FcεR1-KO (right panel) CFs after IgE stimulation were normalized to U6. Three independent experiments were performed. (E) Expression of miR-486a-5p in IgE-stimulated FcεR1-WT and FcεR1-KO CFs at 0, 3, and 24 h measured by qPCR (fold change versus 0 h) in three independent experiments. Results are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, n.s indicates no significance in One-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Antibodies, sources, and dilutions were as follows: rabbit anti-FcεR1 antibody (Cat#10980-1-AP, Proteintech, 1:1000), rabbit anti-αSMA (Cat#ab5694, Abcam, 1:1000), rabbit antiSMAD1 (Cat#6944S, Cell Signaling, 1:1000), rabbit anti-phospho-SMAD1 (Cat#PA5-36771, Invitrogen, 1:1000), rabbit anti-SMAD2 (Cat#12570-1-AP, Proteintech, 1:1000), rabbit anti-phospho-SMAD2 (Cat#18338S, Cell Signaling, 1:1000), rabbit anti-TGFβ1 (Cat#ab179695, Abcam, 1:1000), rabbit antiCollagen I (Cat#PA1-26204, Invitrogen, 1:1000), rabbit anti-Collagen III (Cat#22734-1-AP, Proteintech, 1:1000), rabbit anti-GFP (Cat#ab290, Abcam, 1:1000), and rabbit anti-GAPDH (Cat#10494-1-AP, Proteintech, 1:3000).

Techniques: Expressing

Journal: Theranostics

Article Title: Elevated IgE promotes cardiac fibrosis by suppressing miR-486a-5p.

doi: 10.7150/thno.47845

Figure Lengend Snippet: Figure 5. IgE promotes collagen expression via miR-486a-5p/Smad1 in CFs. (A-B) Representative immunoblot (upper panel) and quantification (lower panel) of SMAD1 protein expression in IgE-stimulated WT (A) and FcεR1-KO (B) CFs at indicated lengths of time (0, 3, 24 h). (C-D) Representative immunoblot (C) and quantification (D) showing protein levels of α-SMA, COL1A1, and COL3A1 in CFs transfected with small interference RNA (siRNA) against Smad1 or scrambled control. (E-F) Representative immunoblot (E) and quantification (F) showing protein levels of α-SMA, COL1A1, and COL3A1 in CFs transfected with Smad1 ORF clone or scrambled control. (G-H) Representative immunoblot (G) and quantification (H) of SMAD1, COL1A1, and COL3A1 protein expression in CFs transfected with miR-486a-5p mimic and/or IgE. (I-J) Immunoblot (I) and quantification (J) showing protein levels of SMAD1, COL1A1, and COL3A1 in CFs transfected with siRNA against Smad1 and/or miR-486a-5p inhibitor. Data are mean ± SD from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s indicates no significant difference in One-way ANOVA with Bonferroni’s post hoc test (A-B), Student’s t-test (D, F) or Two-way ANOVA with Bonferroni’s post hoc test (H, J).

Article Snippet: Antibodies, sources, and dilutions were as follows: rabbit anti-FcεR1 antibody (Cat#10980-1-AP, Proteintech, 1:1000), rabbit anti-αSMA (Cat#ab5694, Abcam, 1:1000), rabbit antiSMAD1 (Cat#6944S, Cell Signaling, 1:1000), rabbit anti-phospho-SMAD1 (Cat#PA5-36771, Invitrogen, 1:1000), rabbit anti-SMAD2 (Cat#12570-1-AP, Proteintech, 1:1000), rabbit anti-phospho-SMAD2 (Cat#18338S, Cell Signaling, 1:1000), rabbit anti-TGFβ1 (Cat#ab179695, Abcam, 1:1000), rabbit antiCollagen I (Cat#PA1-26204, Invitrogen, 1:1000), rabbit anti-Collagen III (Cat#22734-1-AP, Proteintech, 1:1000), rabbit anti-GFP (Cat#ab290, Abcam, 1:1000), and rabbit anti-GAPDH (Cat#10494-1-AP, Proteintech, 1:3000).

Techniques: Expressing, Western Blot, Transfection, Control

Journal: Cell reports

Article Title: Molecular characterization and clinical relevance of metabolic signature subtypes in gastric cancer.

doi: 10.1016/j.celrep.2024.114424

Figure Lengend Snippet: Figure 6. Validation of the metabolic features in an in-house SDPH cohort (A) Metabolic signature profiling of transcriptomic and metabolites profiling of metabolomic among the MSPG and MSC subgroups in in-house SDPH-GC cohort (n = 33). (B) Comparison of the MSPG score, FDX1 expression, cuproptosis, and mismatch repair score among distinct MSC subtypes. (C) Correlations between MSPG score and the known biological signatures curated from Zeng et al. studies with Spearman analysis. The negative and positive correlations are marked with blue and red color. (D) GSVA analysis of representative KEGG pathway among three MSC subtypes in SDPH dataset. Heatmap shows normalized mean enrichment scores of the enriched molecular pathway in three MSC subtypes.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies FDX1 Proteintech Cat# 12592-1-AP; RRID:AB_11182486 LIAS Proteintech Cat# 11577-1-AP; RRID:AB_2135972 DLD Proteintech Cat# 16431-1-AP; RRID:AB_2091888 DLST abcam Cat# ab177934 DLAT Proteintech Cat# 13426-1-AP; RRID:AB_2091774 PDHA1 Proteintech Cat# 18068-1-AP; RRID:AB_2162931 PDHB Proteintech Cat# 14744-1-AP; RRID:AB_2162941 GLS Proteintech Cat# 29519-1-AP; RRID:AB_2918320 IDH1 abways Cat# CY8115 OGDH SAB Cat# 29113 GLS abways Cat# CY5719 TYMS abways Cat# CY7089 ENO2 abways Cat# CY5656 LDHA abways Cat# CY8276 b-actin Proteintech Cat# 20536-1-AP; RRID:AB_10700003 Biological samples

Techniques: Biomarker Discovery, Comparison, Expressing