Structured Review

Qiagen qrt pcr rna
Ro 25–6981 Mechanistic Studies Reveal UPR Modulation in Jimpy (A) <t>qRT-PCR</t> of Plp1 for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. n = 4 technical replicates per sample. (B) qRT-PCR of UPR-related panel for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. For all other probes n = 4 technical replicates per sample. Error bars represent mean ± SD. See also Figure S6 and Table S6 .
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Images

1) Product Images from "Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease"

Article Title: Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease

Journal: Stem Cell Reports

doi: 10.1016/j.stemcr.2018.07.015

Ro 25–6981 Mechanistic Studies Reveal UPR Modulation in Jimpy (A) qRT-PCR of Plp1 for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. n = 4 technical replicates per sample. (B) qRT-PCR of UPR-related panel for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. For all other probes n = 4 technical replicates per sample. Error bars represent mean ± SD. See also Figure S6 and Table S6 .
Figure Legend Snippet: Ro 25–6981 Mechanistic Studies Reveal UPR Modulation in Jimpy (A) qRT-PCR of Plp1 for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. n = 4 technical replicates per sample. (B) qRT-PCR of UPR-related panel for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. For all other probes n = 4 technical replicates per sample. Error bars represent mean ± SD. See also Figure S6 and Table S6 .

Techniques Used: Quantitative RT-PCR

2) Product Images from "Cancer-associated fibroblasts suppress SOX2-induced dysplasia in a lung squamous cancer coculture"

Article Title: Cancer-associated fibroblasts suppress SOX2-induced dysplasia in a lung squamous cancer coculture

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1803718115

CAFs enhance the acinar forming capacity of SOX2oe TUM622 cells and suppress dysplasia. ( A ) Quantification of spheroids formed from Vector and SOX2oe cells as monoculture/coculture with CAFs. Each column represents triplicates. Error bars represent SD. ( B ) CAFs induce the formation of acinar-like structures when in coculture with SOX2oe cells. Both Vector and SOX2oe spheroids become invasive when they are in close proximity with CAFs. (Scales bars, 100 μm.) ( C ) Immunofluorescence of SOX2oe monoculture as well as CAF cocultures stained for Vimentin (cytoplasmic)/p63 (nuclear), E-Cadherin, and ITGA6. Anti-vimentin and anti-p63 antibodies were stained with the same secondary antibody (green). ( D ) Immunofluorescence of SOX2oe monoculture as well as CAF cocultures stained for Vimentin, SOX2, and Phalloidin. (Scale bars, 50 μm.)
Figure Legend Snippet: CAFs enhance the acinar forming capacity of SOX2oe TUM622 cells and suppress dysplasia. ( A ) Quantification of spheroids formed from Vector and SOX2oe cells as monoculture/coculture with CAFs. Each column represents triplicates. Error bars represent SD. ( B ) CAFs induce the formation of acinar-like structures when in coculture with SOX2oe cells. Both Vector and SOX2oe spheroids become invasive when they are in close proximity with CAFs. (Scales bars, 100 μm.) ( C ) Immunofluorescence of SOX2oe monoculture as well as CAF cocultures stained for Vimentin (cytoplasmic)/p63 (nuclear), E-Cadherin, and ITGA6. Anti-vimentin and anti-p63 antibodies were stained with the same secondary antibody (green). ( D ) Immunofluorescence of SOX2oe monoculture as well as CAF cocultures stained for Vimentin, SOX2, and Phalloidin. (Scale bars, 50 μm.)

Techniques Used: Plasmid Preparation, Immunofluorescence, Staining

CAFs increase the number and invasiveness of TUM622 acini. ( A ) Micrograph depicting coculture setups where CAFs are either overlaid or embedded together with TUM622 cells in ECM. After 6 d to 12 d in coculture, TUM622 cells are able to form more and larger acini compared with monoculture, and invade the ECM when in close proximity or direct contact with CAFs. ( B ) Bright-field images of TUM622 3D cultures in the presence or absence of overlaid CAFs after 8 d. (Scale bars, 200 μm.) ( C ) Quantification of acini number in monoculture vs. coculture. Columns represent the average of four replicates performed in technical triplicates, error bars represents SD, and P
Figure Legend Snippet: CAFs increase the number and invasiveness of TUM622 acini. ( A ) Micrograph depicting coculture setups where CAFs are either overlaid or embedded together with TUM622 cells in ECM. After 6 d to 12 d in coculture, TUM622 cells are able to form more and larger acini compared with monoculture, and invade the ECM when in close proximity or direct contact with CAFs. ( B ) Bright-field images of TUM622 3D cultures in the presence or absence of overlaid CAFs after 8 d. (Scale bars, 200 μm.) ( C ) Quantification of acini number in monoculture vs. coculture. Columns represent the average of four replicates performed in technical triplicates, error bars represents SD, and P

Techniques Used:

TME is dominant over the genotype in determining the epithelial architecture of TUM622 cells in vitro. A subpopulation of TUM622 cells has the ability to form acinar-like structures when plated as single cells in ECM culture. These acini are hyperplastic but nevertheless display proper apical−basal cell polarity, similar to hyperplasia observed at the earliest stage of LUSC development. Overexpression of SOX2 in TUM622 cells increases spheroid-forming capacity of TUM622 cells and drives a hyperplastic to dysplastic change in acinar phenotype where apical−basal cell polarity is disrupted and solid spheroids are formed. SOX2oe spheroids also display reduced apoptosis and increased differentiation. Addition of CAFs to TUM622 3D culture enhances acinar formation/growth and promotes invasion toward the CAFs. CAFs could promote the formation of an acinar-like structure in SOX2oe spheroids and induce invasion. Therefore, the key stages of LUSC development, from hyperplasia to dysplasia and eventually invasion, can be observed in this model system.
Figure Legend Snippet: TME is dominant over the genotype in determining the epithelial architecture of TUM622 cells in vitro. A subpopulation of TUM622 cells has the ability to form acinar-like structures when plated as single cells in ECM culture. These acini are hyperplastic but nevertheless display proper apical−basal cell polarity, similar to hyperplasia observed at the earliest stage of LUSC development. Overexpression of SOX2 in TUM622 cells increases spheroid-forming capacity of TUM622 cells and drives a hyperplastic to dysplastic change in acinar phenotype where apical−basal cell polarity is disrupted and solid spheroids are formed. SOX2oe spheroids also display reduced apoptosis and increased differentiation. Addition of CAFs to TUM622 3D culture enhances acinar formation/growth and promotes invasion toward the CAFs. CAFs could promote the formation of an acinar-like structure in SOX2oe spheroids and induce invasion. Therefore, the key stages of LUSC development, from hyperplasia to dysplasia and eventually invasion, can be observed in this model system.

Techniques Used: In Vitro, Over Expression

Cells from TUM622 acini are capable of self-renewal and recapitulate the intratumoral heterogeneity observed in the PDX model and original human tumor. ( A ) Quantification of acinar-like structures formed in an LDA. Each seeding density is plated in triplicate. ( B ) Quantification of acini formed from three serial passages of TUM622 acini in Matrigel. Each passage is plated in triplicate. Error bars represent SD. ( C ) Quantification of in vivo growth of s.c. injected TUM622 acini; x axis represents days after injection, and y axis represents tumor volume (cubic millimeters). Error bars represent SEM. ( D ) Antibody staining of acini with markers of stem/progenitor cells (CXCR4 and SOX2), mesenchyme (Vimentin), epithelial differentiation (Involucrin), apoptosis (CC-3), and proliferation (Ki-67) in green; DAPI (blue), and CDH1 and Phalloidin (red). (Scale bars, 50 μm.) ( E ) Immunohistochemistry staining on sequential sections of TUM622 PDX and the human tumor from which it is derived. Magnification: 40×. Yellow arrow heads point to Vimentin and E-Cadherin double-positive cells. Red arrows point to cells undergoing apoptosis (CC-3−positive), and black arrows point to adjacent Involucrin positive cells that are negative for CC-3. (Scale bars, 60 µm.)
Figure Legend Snippet: Cells from TUM622 acini are capable of self-renewal and recapitulate the intratumoral heterogeneity observed in the PDX model and original human tumor. ( A ) Quantification of acinar-like structures formed in an LDA. Each seeding density is plated in triplicate. ( B ) Quantification of acini formed from three serial passages of TUM622 acini in Matrigel. Each passage is plated in triplicate. Error bars represent SD. ( C ) Quantification of in vivo growth of s.c. injected TUM622 acini; x axis represents days after injection, and y axis represents tumor volume (cubic millimeters). Error bars represent SEM. ( D ) Antibody staining of acini with markers of stem/progenitor cells (CXCR4 and SOX2), mesenchyme (Vimentin), epithelial differentiation (Involucrin), apoptosis (CC-3), and proliferation (Ki-67) in green; DAPI (blue), and CDH1 and Phalloidin (red). (Scale bars, 50 μm.) ( E ) Immunohistochemistry staining on sequential sections of TUM622 PDX and the human tumor from which it is derived. Magnification: 40×. Yellow arrow heads point to Vimentin and E-Cadherin double-positive cells. Red arrows point to cells undergoing apoptosis (CC-3−positive), and black arrows point to adjacent Involucrin positive cells that are negative for CC-3. (Scale bars, 60 µm.)

Techniques Used: In Vivo, Injection, Staining, Immunohistochemistry, Derivative Assay

TUM622 is capable of forming acinar-like structures in 3D ECM. ( A ) Representative bright-field images of spheroids derived from single NSCLC cells in 3D ECM culture between days 8 and 12. ( Top ) Cell lines (TUM622, TUM426, TUM449, and TUM110) are derived from either patient tumors or the corresponding PDX. ( Bottom ) Established cell lines of LUSC (H520), LUAD (HCC2935), and immortalized bronchial epithelial cells (BEAS2B and NHBE). (Scale bars, 50 μm.) ( B ) Time course images of TUM622 cell cultured in Matrigel over a 10-d period. ( Top ) Bright-field images. ( Bottom ) Green is calcein, marking live cells; red is ethidium bromide, marking dead cells. (Scale bars, 100 μm.) ( C ) Quantification of acini number (right y axis, red) and average size of acini (left y axis, blue) plated in triplicate in a 24-well plate over 23 d in culture. Error bars represent SD. ( D ) ( Left ) Immunofluorescence of TUM622 acini stained with apical−basal cell polarity markers Golgi enzyme GM130 (green, apical) and ITGA6 (CD49f, basal, red), ( Right ) in comparison with β-catenin staining (green). (Scale bars, 50 μm.)
Figure Legend Snippet: TUM622 is capable of forming acinar-like structures in 3D ECM. ( A ) Representative bright-field images of spheroids derived from single NSCLC cells in 3D ECM culture between days 8 and 12. ( Top ) Cell lines (TUM622, TUM426, TUM449, and TUM110) are derived from either patient tumors or the corresponding PDX. ( Bottom ) Established cell lines of LUSC (H520), LUAD (HCC2935), and immortalized bronchial epithelial cells (BEAS2B and NHBE). (Scale bars, 50 μm.) ( B ) Time course images of TUM622 cell cultured in Matrigel over a 10-d period. ( Top ) Bright-field images. ( Bottom ) Green is calcein, marking live cells; red is ethidium bromide, marking dead cells. (Scale bars, 100 μm.) ( C ) Quantification of acini number (right y axis, red) and average size of acini (left y axis, blue) plated in triplicate in a 24-well plate over 23 d in culture. Error bars represent SD. ( D ) ( Left ) Immunofluorescence of TUM622 acini stained with apical−basal cell polarity markers Golgi enzyme GM130 (green, apical) and ITGA6 (CD49f, basal, red), ( Right ) in comparison with β-catenin staining (green). (Scale bars, 50 μm.)

Techniques Used: Derivative Assay, Cell Culture, Immunofluorescence, Staining

SOX2 overexpression enhances the spheroid forming capacity of TUM622 cells and induces dysplasia. ( A ) Western blot comparing the expression level of SOX2 in Vector and SOX2oe cells to three LUSC cell lines with no/low (SW900), medium (H2170), and high (H520) levels of SOX2 expression. ( B ) SOX2 expression level in Vector vs. SOX2oe spheroids in 3D culture as quantified by qRT-PCR. Column represents triplicates, and error bars represent SD; P
Figure Legend Snippet: SOX2 overexpression enhances the spheroid forming capacity of TUM622 cells and induces dysplasia. ( A ) Western blot comparing the expression level of SOX2 in Vector and SOX2oe cells to three LUSC cell lines with no/low (SW900), medium (H2170), and high (H520) levels of SOX2 expression. ( B ) SOX2 expression level in Vector vs. SOX2oe spheroids in 3D culture as quantified by qRT-PCR. Column represents triplicates, and error bars represent SD; P

Techniques Used: Over Expression, Western Blot, Expressing, Plasmid Preparation, Quantitative RT-PCR

Gene sets enriched in TUM622 3D vs. 2D culture. ( A ) Top four enriched gene sets identified from TUM622 3D culture (in comparison with TUM622 2D culture) that belong to each of the following categories: key signaling pathways (Wnt, Notch, and Hedgehog signaling), ES cell, polarity/acinar morphogenesis, and prognosis. Horizontal axis represents the −log 10 of FDR value, and the vertical axis represents NES. See the entire list of enriched gene sets corresponding to each category in Dataset S1 . ( B – E ) Enrichment plots of the top two enriched gene sets in each of the categories represented in A .
Figure Legend Snippet: Gene sets enriched in TUM622 3D vs. 2D culture. ( A ) Top four enriched gene sets identified from TUM622 3D culture (in comparison with TUM622 2D culture) that belong to each of the following categories: key signaling pathways (Wnt, Notch, and Hedgehog signaling), ES cell, polarity/acinar morphogenesis, and prognosis. Horizontal axis represents the −log 10 of FDR value, and the vertical axis represents NES. See the entire list of enriched gene sets corresponding to each category in Dataset S1 . ( B – E ) Enrichment plots of the top two enriched gene sets in each of the categories represented in A .

Techniques Used:

3) Product Images from "Faustovirus E12 Transcriptome Analysis Reveals Complex Splicing in Capsid Gene"

Article Title: Faustovirus E12 Transcriptome Analysis Reveals Complex Splicing in Capsid Gene

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02534

Flowchart illustrating the workflow of this study. This flowchart shows the general pipeline of this RNA-seq study, starting from sample preparation and RNA extraction to cDNA sequencing and data analyses. The biological interpretation of expression count was possible through the functional categories clustering of expressed genes.
Figure Legend Snippet: Flowchart illustrating the workflow of this study. This flowchart shows the general pipeline of this RNA-seq study, starting from sample preparation and RNA extraction to cDNA sequencing and data analyses. The biological interpretation of expression count was possible through the functional categories clustering of expressed genes.

Techniques Used: RNA Sequencing Assay, Sample Prep, RNA Extraction, Sequencing, Expressing, Functional Assay

4) Product Images from "Delineating the Dynamic Transcriptome Response of mRNA and microRNA during Zebrafish Heart Regeneration"

Article Title: Delineating the Dynamic Transcriptome Response of mRNA and microRNA during Zebrafish Heart Regeneration

Journal: Biomolecules

doi: 10.3390/biom9010011

( A ) Experimental design of the H9c2 cell line. Both mRNA and miRNA were sequenced at 4 different time points that display a differentiation into a CM-like phenotype and a reduction of the proliferative capability. ( B ) Proportions of the significant down-regulated genes (d5 vs. undiff; FDR
Figure Legend Snippet: ( A ) Experimental design of the H9c2 cell line. Both mRNA and miRNA were sequenced at 4 different time points that display a differentiation into a CM-like phenotype and a reduction of the proliferative capability. ( B ) Proportions of the significant down-regulated genes (d5 vs. undiff; FDR

Techniques Used:

5) Product Images from "Constitutive Changes in Circulating Follicular Helper T Cells and Their Subsets in Patients with Graves' Disease"

Article Title: Constitutive Changes in Circulating Follicular Helper T Cells and Their Subsets in Patients with Graves' Disease

Journal: Journal of Immunology Research

doi: 10.1155/2018/8972572

mRNA expression in human CD4 + T cells from GD patients. The expression of several transcription factors (Bcl-6, T-bet, GATA-3, and ROR γ t) in peripheral CD4 + T cells from the 9 GD patients and 12 HC was detected with a real-time PCR assay. (a) Relative expression levels of Bcl-6 mRNA; (b) relative expression levels of T-bet mRNA; (c) relative expression levels of GATA-3 mRNA; (d) relative expression levels of ROR γ t mRNA. ∗ p
Figure Legend Snippet: mRNA expression in human CD4 + T cells from GD patients. The expression of several transcription factors (Bcl-6, T-bet, GATA-3, and ROR γ t) in peripheral CD4 + T cells from the 9 GD patients and 12 HC was detected with a real-time PCR assay. (a) Relative expression levels of Bcl-6 mRNA; (b) relative expression levels of T-bet mRNA; (c) relative expression levels of GATA-3 mRNA; (d) relative expression levels of ROR γ t mRNA. ∗ p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

6) Product Images from "Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor Roquin2"

Article Title: Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor Roquin2

Journal: Nature cell biology

doi: 10.1038/s41556-018-0084-5

KLHL6 is a BCR/NF-κB target gene that links Roquin2 degradation to BCR signaling (a) Immunoblot analysis of whole cell lysates from OCI-LY10, U2932, and HBL1 cells stimulated with 10 μg/ml F(ab′)2-IgM for 3 and 6 hours (left panel). A low exposure (l.e.) and high exposure (h.e.) are shown. Right panel shows level of KLHL6 mRNA analyzed by qPCR. The value for the PCR product from U2932 was set as 1. A representative graph from two independent experiments is shown. (b) Immunoblot analysis of whole cell lysates from U2932 cells treated with 10 μg/ml of F(ab′)2-IgM for the indicated times. Where indicated, cells were pre-treated with 5μM MLN4924 for 1 hour. (c) Analysis of level of KLHL6 mRNA by qPCR in U2932 cells treated with 10 μg/ml of F(ab′)2-IgM for the indicated times. A representative graph from two independent experiments is shown. The value for PCR product without treatment was set as 1. (d) Visualization of ChIP-seq peaks using the University of California Santa Cruz (UCSD) Genome browser (GEO Series accession GSE55105). RPM, reads per million mapped. (e) Same as in (c) except that U2932 cells were treated with DMSO, 10μM of IKK inhibitor (IKK-16) or 5μM of BTK inhibitor (Ibrutinib) for 6 hours. The value for PCR product present without treatment (DMSO) was set as 100% (mean±s.d., n=3 independent experiments, one-way ANOVA, *** P value≤0.001; ****P value≤0.0001). The right panel shows immunoblot analysis of whole cell lysates for the indicated proteins. (f) Immunoblot analysis of whole cell lysates from U2932 KLHL6 +/+ and KLHL6 −/− (clone-derived) cells treated with increasing concentrations of F(ab′)2-IgM for 6 hours. (g) Immunoblot analysis of whole cell lysates from U2932 cells stably expressing HA-Roquin2(WT) or HA-Roquin2(Y691F) treated with F(ab′)2-IgM for 6 hours. Unprocessed original scan of immunoblots for (a,b,e,f,g) are shown in Supplementary Fig. 8 , and source data for (a,c) and statistical source data and exact P values for (e) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.
Figure Legend Snippet: KLHL6 is a BCR/NF-κB target gene that links Roquin2 degradation to BCR signaling (a) Immunoblot analysis of whole cell lysates from OCI-LY10, U2932, and HBL1 cells stimulated with 10 μg/ml F(ab′)2-IgM for 3 and 6 hours (left panel). A low exposure (l.e.) and high exposure (h.e.) are shown. Right panel shows level of KLHL6 mRNA analyzed by qPCR. The value for the PCR product from U2932 was set as 1. A representative graph from two independent experiments is shown. (b) Immunoblot analysis of whole cell lysates from U2932 cells treated with 10 μg/ml of F(ab′)2-IgM for the indicated times. Where indicated, cells were pre-treated with 5μM MLN4924 for 1 hour. (c) Analysis of level of KLHL6 mRNA by qPCR in U2932 cells treated with 10 μg/ml of F(ab′)2-IgM for the indicated times. A representative graph from two independent experiments is shown. The value for PCR product without treatment was set as 1. (d) Visualization of ChIP-seq peaks using the University of California Santa Cruz (UCSD) Genome browser (GEO Series accession GSE55105). RPM, reads per million mapped. (e) Same as in (c) except that U2932 cells were treated with DMSO, 10μM of IKK inhibitor (IKK-16) or 5μM of BTK inhibitor (Ibrutinib) for 6 hours. The value for PCR product present without treatment (DMSO) was set as 100% (mean±s.d., n=3 independent experiments, one-way ANOVA, *** P value≤0.001; ****P value≤0.0001). The right panel shows immunoblot analysis of whole cell lysates for the indicated proteins. (f) Immunoblot analysis of whole cell lysates from U2932 KLHL6 +/+ and KLHL6 −/− (clone-derived) cells treated with increasing concentrations of F(ab′)2-IgM for 6 hours. (g) Immunoblot analysis of whole cell lysates from U2932 cells stably expressing HA-Roquin2(WT) or HA-Roquin2(Y691F) treated with F(ab′)2-IgM for 6 hours. Unprocessed original scan of immunoblots for (a,b,e,f,g) are shown in Supplementary Fig. 8 , and source data for (a,c) and statistical source data and exact P values for (e) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.

Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Derivative Assay, Stable Transfection, Expressing, Western Blot

KLHL6-Roquin2 axis controls NF-κB activation (a) Immunoblot analysis of whole cell lysates from U2932 cells expressing HA-Roquin2(WT) or HA-Roquin2(Y691F) treated with 10μg/ml of F(ab′)2-IgM for the indicated times. (b) Immunoblot analysis of whole cell lysates from U2932 KLHL6 +/+ or KLHL6 −/− cells (clone-derived) treated with 10μg/ml of F(ab′)2-IgM for the indicated times. (c) Immunoblot analysis of whole cell lysates from HBL1 cells electroporated with indicated siRNAs and treated as in (a) for the indicated times. (d) Immunoblot analysis of whole cell lysates from U2932 KLHL6 +/+ or KLHL6 −/− cells (clo ne-derived) treated with 10μg/ml of F(ab′)2-IgM for the indicated times. (e) Immunoblot analysis of fractionated U2932 KLHL6 +/+ or KLHL6 −/− (clone-derived) cells. (f) RelA ChIP-qPCR for the NFBKIA promoter in U2932 KLHL6 +/+ , KLHL6 −/− or KLHL6 −/− cells infected with indicated shRNAs. Data are displayed as fold enrichment relative to IgG control. A representative graph from two independent experiments is shown. (g) Overlap of BTB-associated mutations of KLHL6 with TNFAIP3 alterations in DLBCLs. Top panel shows tumors sequenced at UNMC 6 and DCI 7 (n=1175) with deleterious mutations of KLHL6 in the BTB-domain and TNFAIP3 mutations. In the bottom panel, the TNFAIP3 subset is shown as biallelic and monoallelic deletions. (h) Cell counts of GFP-sorted RCK8 cells expressing Cas9, the indicated gRNAs and a GFP marker. Cells were grown in media containing 1μg/ml of F(ab′)2-IgM (mean±s.d., n=3 independent experiments, two-way ANOVA, n.s, not significant). Right panel shows immunoblot analysis of whole cell lysates. (i) Apoptosis analysis of cells from (h) is shown (mean±s.d., n=3 independent experiments, one-way ANOVA, n.s, not significant). (j) The percentage of GFP + and AnnexinV + HBL1 and HLY-1 cells expressing the indicated shRNAs is shown (mean±s.d., n=3 independent experiments, one-way ANOVA, *P value≤0.05; *** P value≤0.001, n.s, not significant). (k) Model of KLHL6-Roquin2 axis in ABC-DLBCL. Unprocessed original scan of immunoblots for (a,b,c,d,e,h) are shown in Supplementary Fig. 8 , and source data for (f) and statistical source data for (h,i,j) and exact P values for (j) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.
Figure Legend Snippet: KLHL6-Roquin2 axis controls NF-κB activation (a) Immunoblot analysis of whole cell lysates from U2932 cells expressing HA-Roquin2(WT) or HA-Roquin2(Y691F) treated with 10μg/ml of F(ab′)2-IgM for the indicated times. (b) Immunoblot analysis of whole cell lysates from U2932 KLHL6 +/+ or KLHL6 −/− cells (clone-derived) treated with 10μg/ml of F(ab′)2-IgM for the indicated times. (c) Immunoblot analysis of whole cell lysates from HBL1 cells electroporated with indicated siRNAs and treated as in (a) for the indicated times. (d) Immunoblot analysis of whole cell lysates from U2932 KLHL6 +/+ or KLHL6 −/− cells (clo ne-derived) treated with 10μg/ml of F(ab′)2-IgM for the indicated times. (e) Immunoblot analysis of fractionated U2932 KLHL6 +/+ or KLHL6 −/− (clone-derived) cells. (f) RelA ChIP-qPCR for the NFBKIA promoter in U2932 KLHL6 +/+ , KLHL6 −/− or KLHL6 −/− cells infected with indicated shRNAs. Data are displayed as fold enrichment relative to IgG control. A representative graph from two independent experiments is shown. (g) Overlap of BTB-associated mutations of KLHL6 with TNFAIP3 alterations in DLBCLs. Top panel shows tumors sequenced at UNMC 6 and DCI 7 (n=1175) with deleterious mutations of KLHL6 in the BTB-domain and TNFAIP3 mutations. In the bottom panel, the TNFAIP3 subset is shown as biallelic and monoallelic deletions. (h) Cell counts of GFP-sorted RCK8 cells expressing Cas9, the indicated gRNAs and a GFP marker. Cells were grown in media containing 1μg/ml of F(ab′)2-IgM (mean±s.d., n=3 independent experiments, two-way ANOVA, n.s, not significant). Right panel shows immunoblot analysis of whole cell lysates. (i) Apoptosis analysis of cells from (h) is shown (mean±s.d., n=3 independent experiments, one-way ANOVA, n.s, not significant). (j) The percentage of GFP + and AnnexinV + HBL1 and HLY-1 cells expressing the indicated shRNAs is shown (mean±s.d., n=3 independent experiments, one-way ANOVA, *P value≤0.05; *** P value≤0.001, n.s, not significant). (k) Model of KLHL6-Roquin2 axis in ABC-DLBCL. Unprocessed original scan of immunoblots for (a,b,c,d,e,h) are shown in Supplementary Fig. 8 , and source data for (f) and statistical source data for (h,i,j) and exact P values for (j) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.

Techniques Used: Activation Assay, Expressing, Derivative Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Infection, Marker, Western Blot

KLHL6 interacts and promotes ubiquitylation and degradation of Roquin2 (a) Proteomic analysis of KLHL6 immunoprecipitations. Spectral counts for Roquin2 proteins are shown. EV, empty vector. The analysis was performed once in two different cell lines (HEK293T and ARP-1). (b) Immunoblot analysis of immunoprecipitated FLAG-F-box proteins (FBPs) or BTB proteins (BTBPs) in HEK293T cells. Lane 1 shows whole cell lysates (WCL) from cells transfected with an empty vector. (c) Immunoblot analysis of immunoprecipitated FLAG-Roquin1 or FLAG-Roquin2 in HEK293T cells. (d) Immunoblot analysis of human DLBCL cell lysates. A low exposure (l.e.) and high exposure (h.e.) are shown. A representative blot from two independent experiments is shown. (e) Immunoblot analysis of whole cell lysates from OCI-LY10 cells electroporated with indicated siRNAs and treated with cycloheximide (CHX). Quantification and statistical analysis is shown in Fig. S2d . (f) KLHL6 +/+ and KLHL6 −/− U2932 cells(clone-derived) were processed as in (e). A low exposure (l.e.) and high exposure (h.e.) are shown. Quantification and statistical analysis is shown in Fig. S2d . (g) Schematic representation of KLHL6 protein displaying endogenous mutations in VAL and SUDHL10 (top panel). Bottom panel shows immunoblot analysis from immunoprecipitated FLAG-tagged KLHL6 wild-type (WT), KLHL6 mutants (L24R, A25E, N60T, and T72R), or empty vector (EV) in HEK293T cells. (h) Immunoblot analysis of whole cell lysates from VAL and SUDHL10 cells electroporated with siRNA scramble (siCTRL) or targeting KLHL6 (siKLHL6). (i) Analysis of level of KLHL6 mRNA by quantitative PCR(qPCR). The value for the PCR product from U2932 cells was set as 1. A representative graph from two independent experiments is shown. (j) Immunoblot analysis of whole cell lysates from VAL and SUDHL10 cells stably expressing KLHL6 under a doxycycline (DOX) inducible promoter with a puryomycin cassette after 12h of DOX treatment. (k) In vitro ubiquitylation reaction of immunopurified FLAG-KLHL6 and HA-Roquin2. Unprocessed original scan of immunoblots for (b,c,d,e,f,g,h,j,k) are shown in Supplementary Fig. 8 , and source data for (i) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.
Figure Legend Snippet: KLHL6 interacts and promotes ubiquitylation and degradation of Roquin2 (a) Proteomic analysis of KLHL6 immunoprecipitations. Spectral counts for Roquin2 proteins are shown. EV, empty vector. The analysis was performed once in two different cell lines (HEK293T and ARP-1). (b) Immunoblot analysis of immunoprecipitated FLAG-F-box proteins (FBPs) or BTB proteins (BTBPs) in HEK293T cells. Lane 1 shows whole cell lysates (WCL) from cells transfected with an empty vector. (c) Immunoblot analysis of immunoprecipitated FLAG-Roquin1 or FLAG-Roquin2 in HEK293T cells. (d) Immunoblot analysis of human DLBCL cell lysates. A low exposure (l.e.) and high exposure (h.e.) are shown. A representative blot from two independent experiments is shown. (e) Immunoblot analysis of whole cell lysates from OCI-LY10 cells electroporated with indicated siRNAs and treated with cycloheximide (CHX). Quantification and statistical analysis is shown in Fig. S2d . (f) KLHL6 +/+ and KLHL6 −/− U2932 cells(clone-derived) were processed as in (e). A low exposure (l.e.) and high exposure (h.e.) are shown. Quantification and statistical analysis is shown in Fig. S2d . (g) Schematic representation of KLHL6 protein displaying endogenous mutations in VAL and SUDHL10 (top panel). Bottom panel shows immunoblot analysis from immunoprecipitated FLAG-tagged KLHL6 wild-type (WT), KLHL6 mutants (L24R, A25E, N60T, and T72R), or empty vector (EV) in HEK293T cells. (h) Immunoblot analysis of whole cell lysates from VAL and SUDHL10 cells electroporated with siRNA scramble (siCTRL) or targeting KLHL6 (siKLHL6). (i) Analysis of level of KLHL6 mRNA by quantitative PCR(qPCR). The value for the PCR product from U2932 cells was set as 1. A representative graph from two independent experiments is shown. (j) Immunoblot analysis of whole cell lysates from VAL and SUDHL10 cells stably expressing KLHL6 under a doxycycline (DOX) inducible promoter with a puryomycin cassette after 12h of DOX treatment. (k) In vitro ubiquitylation reaction of immunopurified FLAG-KLHL6 and HA-Roquin2. Unprocessed original scan of immunoblots for (b,c,d,e,f,g,h,j,k) are shown in Supplementary Fig. 8 , and source data for (i) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.

Techniques Used: Plasmid Preparation, Immunoprecipitation, Transfection, Derivative Assay, Polymerase Chain Reaction, Stable Transfection, Expressing, In Vitro, Western Blot

KLHL6 functions as a tumor suppressor in ABC-DLBCL by regulating its growth and survival (a) Kaplan–Meier analysis based on gene expression data for ABC-DLBCL tumors (GSE10846, GSE34171 and GSE3131239-41) is shown (n=367 patients). Censored subjects are indicated on the Kaplan-Meier cure as tick mark. Statistical analysis was performed using the Log-rank (Mantel-Cox), two-sided test, 95% confidence interval. (b) Cell counts of U2932-, OCI-LY10- and TMD8-Cas9 cells expressing the indicated gRNAs and carrying a puromycin cassette (mean±s.d., n=3 independent experiments, two-way ANOVA, *P value≤0.05; **P value≤0.01; *** P value≤0.001; ****P value≤0.0001) (left panel). Cells were grown in media containing 1μg/ml (U2932- and OCI-LY10) or 4μg/ml (TMD8) of F(ab′)2-IgM. Right panel shows immunoblot analysis of whole cell lysates. (c) Apoptosis analysis of U2932-, OCI-LY10-, and TMD8-Cas9 cells expressing the indicated gRNAs and carrying a GFP marker. Cells were grown as in (b) . Apoptosis was quantified on GFP + and Annexin V + cells (mean±s.d., n=3 independent experiments, one-way ANOVA, **P value≤0.01; *** P value≤0.001). (d) Left panel shows cell counts of GFP-sorted U2932 KLHL6 −/− (clone-derived) cells expressing empty vector (EV), KLHL6(WT) or BTB-mutants (L65P, S94I and F97L) and carrying a GFP marker (mean±s.d., n=3 independent experiments, two-way ANOVA, **P value≤0.01; *** P value≤0.001; ****P value≤0.0001). Right panel shows the immunoblot analysis of whole cell lysates. (e) Xenograft experiments with GFP-sorted U2932 KLHL6 −/− cells expressing an empty vector (EV), KLHL6(WT) or KLHL6(S94I) and carrying a GFP marker. Top panel shows the tumors at the experimental endpoint. Tumor volume (mean±s.d., n=5 mice per group, one-way ANOVA, *P value≤0.05; n.s, not significant) and tumor weight (mean±s.d., n=5 mice per group, one-tailed t-test, *P value ≤0.05; **P value ≤0.01; n.s, not significant) are shown in the bottom left and right, respectively. Unprocessed original scan of immunoblots for (b,d) are shown in Supplementary Fig. 8 , and statistical source data and exact P values for (a,b,c,d,e) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.
Figure Legend Snippet: KLHL6 functions as a tumor suppressor in ABC-DLBCL by regulating its growth and survival (a) Kaplan–Meier analysis based on gene expression data for ABC-DLBCL tumors (GSE10846, GSE34171 and GSE3131239-41) is shown (n=367 patients). Censored subjects are indicated on the Kaplan-Meier cure as tick mark. Statistical analysis was performed using the Log-rank (Mantel-Cox), two-sided test, 95% confidence interval. (b) Cell counts of U2932-, OCI-LY10- and TMD8-Cas9 cells expressing the indicated gRNAs and carrying a puromycin cassette (mean±s.d., n=3 independent experiments, two-way ANOVA, *P value≤0.05; **P value≤0.01; *** P value≤0.001; ****P value≤0.0001) (left panel). Cells were grown in media containing 1μg/ml (U2932- and OCI-LY10) or 4μg/ml (TMD8) of F(ab′)2-IgM. Right panel shows immunoblot analysis of whole cell lysates. (c) Apoptosis analysis of U2932-, OCI-LY10-, and TMD8-Cas9 cells expressing the indicated gRNAs and carrying a GFP marker. Cells were grown as in (b) . Apoptosis was quantified on GFP + and Annexin V + cells (mean±s.d., n=3 independent experiments, one-way ANOVA, **P value≤0.01; *** P value≤0.001). (d) Left panel shows cell counts of GFP-sorted U2932 KLHL6 −/− (clone-derived) cells expressing empty vector (EV), KLHL6(WT) or BTB-mutants (L65P, S94I and F97L) and carrying a GFP marker (mean±s.d., n=3 independent experiments, two-way ANOVA, **P value≤0.01; *** P value≤0.001; ****P value≤0.0001). Right panel shows the immunoblot analysis of whole cell lysates. (e) Xenograft experiments with GFP-sorted U2932 KLHL6 −/− cells expressing an empty vector (EV), KLHL6(WT) or KLHL6(S94I) and carrying a GFP marker. Top panel shows the tumors at the experimental endpoint. Tumor volume (mean±s.d., n=5 mice per group, one-way ANOVA, *P value≤0.05; n.s, not significant) and tumor weight (mean±s.d., n=5 mice per group, one-tailed t-test, *P value ≤0.05; **P value ≤0.01; n.s, not significant) are shown in the bottom left and right, respectively. Unprocessed original scan of immunoblots for (b,d) are shown in Supplementary Fig. 8 , and statistical source data and exact P values for (a,b,c,d,e) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.

Techniques Used: Expressing, Marker, Derivative Assay, Plasmid Preparation, Mouse Assay, One-tailed Test, Western Blot

A non-degradable Roquin2 mutant phenocopies loss of KLHL6 (a) Left panel shows immunoblot analysis of immunoprecipitated FLAG-tagged Roquin2 wild type (WT) or mutants in HEK293T cells stably expressing KLHL6. EV, Empty vector. Right panel shows a schematic representation of Roquin2 mutants. Roquin2 mutants that interact (+) or do not interact (-) with KLHL6 are shown. A representative blot from two independent experiments is shown. Asterisk indicates non-specific bands. (b–d) Same as in (a). (e) Immunoblot analysis of immunoprecipitated FLAG-tagged Roquin2 wild type (WT) or mutants, as indicated, in HEK293T cells stably expressing KLHL6. EV, Empty vector. (f) Immunoblot analysis of whole cell lysates from a DLBCL cell line, BJAB, retrovirally transduced with cDNAs encoding Roquin2(WT) or Roquin2(Y691F) carrying a puromycin cassette. Cells were treated with cycloheximide (CHX) for the indicated times. Quantification and statistical analysis is shown in Fig. S4e . (g) Top panel shows tumor volume from subcutaneously injected NSG mice with U2932 cells stably expressing retroviruses encoding HA-Roquin2(WT) or HA-Roquin2(Y691F) carrying a puromycin cassette (mean±s.d., n=3 mice per group, two-way ANOVA, *P value≤0.05; **P value≤0.01; *** P value≤0.001; ****P value≤0.0001). Bottom panel shows tumor weight (mean±s.d., n=3 mice per group, two-tailed t-test, *** P value≤0.001). (h) Cell counts of GFP-sorted U2932 (left panel) or OCI-LY10 (right panel) KLHL6 +/+ and KLHL6 −/− (clone-derived) cells infected with scramble shRNA(shCTRL) or shRNA targeting Roquin2 (ShRoquin2#1 or #2) carrying a GFP marker. GFP + cells were grown in media containing 1μg/ml of F(ab′)2-IgM. (mean±s.d., n=3 independent experiments, two-way ANOVA, ****P value≤0.0001). Unprocessed original scan of immunoblots for (a,b,c,d,e,f) are shown in Supplementary Fig. 8 , and statistical source data and exact P values for (g,h) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.
Figure Legend Snippet: A non-degradable Roquin2 mutant phenocopies loss of KLHL6 (a) Left panel shows immunoblot analysis of immunoprecipitated FLAG-tagged Roquin2 wild type (WT) or mutants in HEK293T cells stably expressing KLHL6. EV, Empty vector. Right panel shows a schematic representation of Roquin2 mutants. Roquin2 mutants that interact (+) or do not interact (-) with KLHL6 are shown. A representative blot from two independent experiments is shown. Asterisk indicates non-specific bands. (b–d) Same as in (a). (e) Immunoblot analysis of immunoprecipitated FLAG-tagged Roquin2 wild type (WT) or mutants, as indicated, in HEK293T cells stably expressing KLHL6. EV, Empty vector. (f) Immunoblot analysis of whole cell lysates from a DLBCL cell line, BJAB, retrovirally transduced with cDNAs encoding Roquin2(WT) or Roquin2(Y691F) carrying a puromycin cassette. Cells were treated with cycloheximide (CHX) for the indicated times. Quantification and statistical analysis is shown in Fig. S4e . (g) Top panel shows tumor volume from subcutaneously injected NSG mice with U2932 cells stably expressing retroviruses encoding HA-Roquin2(WT) or HA-Roquin2(Y691F) carrying a puromycin cassette (mean±s.d., n=3 mice per group, two-way ANOVA, *P value≤0.05; **P value≤0.01; *** P value≤0.001; ****P value≤0.0001). Bottom panel shows tumor weight (mean±s.d., n=3 mice per group, two-tailed t-test, *** P value≤0.001). (h) Cell counts of GFP-sorted U2932 (left panel) or OCI-LY10 (right panel) KLHL6 +/+ and KLHL6 −/− (clone-derived) cells infected with scramble shRNA(shCTRL) or shRNA targeting Roquin2 (ShRoquin2#1 or #2) carrying a GFP marker. GFP + cells were grown in media containing 1μg/ml of F(ab′)2-IgM. (mean±s.d., n=3 independent experiments, two-way ANOVA, ****P value≤0.0001). Unprocessed original scan of immunoblots for (a,b,c,d,e,f) are shown in Supplementary Fig. 8 , and statistical source data and exact P values for (g,h) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.

Techniques Used: Mutagenesis, Immunoprecipitation, Stable Transfection, Expressing, Plasmid Preparation, Transduction, Injection, Mouse Assay, Two Tailed Test, Derivative Assay, Infection, shRNA, Marker, Western Blot

Stabilization of Roquin2 down-regulates BCR responsive genes (a) Representative image of U2932 cells expressing HA-Roquin2(WT), HA-Roquin2(Y691F) or HA-Roquin2(Y691FΔROQ) with puromycin cassette plated into a matrigel (Left panel). Middle panel shows immunoblot analysis of whole cell lysates and right panel shows cell counts from the matrigel (mean±s.d., n=4 independent experiments, one-way ANOVA, ****P value≤0.0001, n.s, not significant). Scale bar 150μm. (b) Volcano plot (left panel) showing down-regulated mRNAs (blue) in U2932 cells expressing Roquin2(Y691F) vs Roquin2(WT) upon 12 hours treatment with 10 μg/ml of F(ab′)2-IgM [log2(fold-change)
Figure Legend Snippet: Stabilization of Roquin2 down-regulates BCR responsive genes (a) Representative image of U2932 cells expressing HA-Roquin2(WT), HA-Roquin2(Y691F) or HA-Roquin2(Y691FΔROQ) with puromycin cassette plated into a matrigel (Left panel). Middle panel shows immunoblot analysis of whole cell lysates and right panel shows cell counts from the matrigel (mean±s.d., n=4 independent experiments, one-way ANOVA, ****P value≤0.0001, n.s, not significant). Scale bar 150μm. (b) Volcano plot (left panel) showing down-regulated mRNAs (blue) in U2932 cells expressing Roquin2(Y691F) vs Roquin2(WT) upon 12 hours treatment with 10 μg/ml of F(ab′)2-IgM [log2(fold-change)

Techniques Used: Expressing

7) Product Images from "Host suppression of quorum sensing during catheter-associated urinary tract infections"

Article Title: Host suppression of quorum sensing during catheter-associated urinary tract infections

Journal: Nature Communications

doi: 10.1038/s41467-018-06882-y

Urine and urea suppress quorum-regulated phenotypes. P. aeruginosa grown in 1× phosphate-buffered saline + 1% tryptone (PBS-T) supplemented with increasing concentrations of human urine was quantified for a zone of clearance around bacterial colony grown on milk plates, b pyocyanin (red bars) and rhamnolipid (blue bars) production. P. aeruginosa grown in LB supplemented with increasing amounts of urea was quantified for c zone of clearance around colonies grown on milk plates, d pyocyanin (red bars) and rhamnolipid (blue bars) production. e Quantification of pyocyanin produced by P. aeruginosa after 24-h growth in the presence or absence of 0.5 M urea followed by 16-h subculture growth in the presence or absence of 0.5 M urea. Data represent the mean and standard deviation of at least three independent replicates and were analyzed by unpaired t -test using GraphPad Prism software. Asterisk (*, **, and ***) indicates p
Figure Legend Snippet: Urine and urea suppress quorum-regulated phenotypes. P. aeruginosa grown in 1× phosphate-buffered saline + 1% tryptone (PBS-T) supplemented with increasing concentrations of human urine was quantified for a zone of clearance around bacterial colony grown on milk plates, b pyocyanin (red bars) and rhamnolipid (blue bars) production. P. aeruginosa grown in LB supplemented with increasing amounts of urea was quantified for c zone of clearance around colonies grown on milk plates, d pyocyanin (red bars) and rhamnolipid (blue bars) production. e Quantification of pyocyanin produced by P. aeruginosa after 24-h growth in the presence or absence of 0.5 M urea followed by 16-h subculture growth in the presence or absence of 0.5 M urea. Data represent the mean and standard deviation of at least three independent replicates and were analyzed by unpaired t -test using GraphPad Prism software. Asterisk (*, **, and ***) indicates p

Techniques Used: Produced, Standard Deviation, Software

8) Product Images from "Hedgehog signaling has a protective effect in glucocorticoid-induced mouse neonatal brain injury through an 11?HSD2-dependent mechanism"

Article Title: Hedgehog signaling has a protective effect in glucocorticoid-induced mouse neonatal brain injury through an 11?HSD2-dependent mechanism

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI36376

Shh signaling upregulates transcription of 11 β HSD2 in vitro and in vivo. ( A ) Whole RNA was isolated from WT and Math1cre,SmoM2 CGNP cultures treated with vehicle, Shh, or Shh plus 40 μM Dex for 24 h. Quantitative PCR analysis showed that Shh treatment potently induced the Shh targets N-myc and Gli1 as well as 11 β HSD2 expression in WT CGNP cultures. Math1cre,SmoM2 CGNPs showed increased expression of N-myc , Gli1 , and 11 β HSD2 in both vehicle and Shh groups and was unchanged after Dex treatment. 11 β HSD1 expression was below detectable levels. ( B ) In vivo, N-myc , Gli1 , and 11 β HSD2 levels were upregulated in the Math1cre,SmoM2 cerebellum (CB). ( C ) In situ hybridization showing specific expression of 11 β HSD2 in the EGL of the P7 WT and Math1cre,SmoM2 cerebellum. Original magnification, ×400.
Figure Legend Snippet: Shh signaling upregulates transcription of 11 β HSD2 in vitro and in vivo. ( A ) Whole RNA was isolated from WT and Math1cre,SmoM2 CGNP cultures treated with vehicle, Shh, or Shh plus 40 μM Dex for 24 h. Quantitative PCR analysis showed that Shh treatment potently induced the Shh targets N-myc and Gli1 as well as 11 β HSD2 expression in WT CGNP cultures. Math1cre,SmoM2 CGNPs showed increased expression of N-myc , Gli1 , and 11 β HSD2 in both vehicle and Shh groups and was unchanged after Dex treatment. 11 β HSD1 expression was below detectable levels. ( B ) In vivo, N-myc , Gli1 , and 11 β HSD2 levels were upregulated in the Math1cre,SmoM2 cerebellum (CB). ( C ) In situ hybridization showing specific expression of 11 β HSD2 in the EGL of the P7 WT and Math1cre,SmoM2 cerebellum. Original magnification, ×400.

Techniques Used: In Vitro, In Vivo, Isolation, Real-time Polymerase Chain Reaction, Expressing, In Situ Hybridization

9) Product Images from "Phosphorylated and sumoylation-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression"

Article Title: Phosphorylated and sumoylation-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression

Journal: Breast Cancer Research : BCR

doi: 10.1186/bcr3211

Phosphorylation of PR Ser294 drives SUMO-deficient PR gene expression and promoter selectivity in MCF-7 and T47D cells . ( A ) Relative expression level (copy number) of PR target genes in breast cancer patient cohorts. ( B ) Relative gene expression levels of selected PR target genes in MCF-7 cells stably expressing either empty vector (PR-null), WT or SUMO-deficient K388R PRs. Cells were co-treated with progestin R5020 and/or antiprogestin RU486 for six hours and mRNA levels were measured using RT-qPCR (see Materials and methods). ( C ) Relative gene expression levels of the same PR target genes (as in parts A-B) were measured using RT-qPCR in five vector-matched T47D cell lines stably expressing PRs: empty vector (null), wild-type (WT) PR, K388R mutant (KR) PR, S294A mutant (SA) PR, and K388R and S294A double mutant (KRSA) PR. Cells were treated with R5020 for six hours. ( D ) T47D cells expressing WT PR were treated cells with epidermal growth factor (EGF) for two days and treated with R5020 for 3, 24, or 48 hours. Relative MAP1A and RGS2 mRNA levels were measured using RT-qPCR. ( E ) Parental T47Dco cells were pretreated with EGF for 20 minutes prior to 24 hours of R5020 treatment. Relative RGS2 mRNA levels were measured by RT-qPCR. Data are represented as mean of n = 3 +/- SD and significance calculated using Student's t-test. n, number; PR, progesterone receptor; SD, standard deviation; SUMO, small ubiquitin-like modifier; WT, wild type.
Figure Legend Snippet: Phosphorylation of PR Ser294 drives SUMO-deficient PR gene expression and promoter selectivity in MCF-7 and T47D cells . ( A ) Relative expression level (copy number) of PR target genes in breast cancer patient cohorts. ( B ) Relative gene expression levels of selected PR target genes in MCF-7 cells stably expressing either empty vector (PR-null), WT or SUMO-deficient K388R PRs. Cells were co-treated with progestin R5020 and/or antiprogestin RU486 for six hours and mRNA levels were measured using RT-qPCR (see Materials and methods). ( C ) Relative gene expression levels of the same PR target genes (as in parts A-B) were measured using RT-qPCR in five vector-matched T47D cell lines stably expressing PRs: empty vector (null), wild-type (WT) PR, K388R mutant (KR) PR, S294A mutant (SA) PR, and K388R and S294A double mutant (KRSA) PR. Cells were treated with R5020 for six hours. ( D ) T47D cells expressing WT PR were treated cells with epidermal growth factor (EGF) for two days and treated with R5020 for 3, 24, or 48 hours. Relative MAP1A and RGS2 mRNA levels were measured using RT-qPCR. ( E ) Parental T47Dco cells were pretreated with EGF for 20 minutes prior to 24 hours of R5020 treatment. Relative RGS2 mRNA levels were measured by RT-qPCR. Data are represented as mean of n = 3 +/- SD and significance calculated using Student's t-test. n, number; PR, progesterone receptor; SD, standard deviation; SUMO, small ubiquitin-like modifier; WT, wild type.

Techniques Used: Expressing, Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Mutagenesis, Standard Deviation

The SUMO-deficient PR gene expression signature is associated with HER2-positive human breast tumors and predicts reduced patient survival . ( A ) Normalized gene expression levels (for genes in our LD KR > WT gene signature) are presented for each tumor in the patient cohort [ 64 ], organized by ERBB2 status. ( B ) Gene expression levels were measured by RT-qPCR for CHN2 and RGS2 (both upregulated by SUMO-deficient PR, and members of the LD KR > WT gene signature) and the control gene ACOT6 (equally upregulated by both WT and KR receptors) in BT-474 human breast cancer cells. Cells were pre-treated with MEK kinase inhibitor U0126 prior to progestin or antiprogestin co-treatment. Protein levels were evaluated by western blotting for total PR, PR Ser294 phosphorylation, total ERK1/2, and ERK1/2 phosphorylation. ( C ) Kaplan-Meier survival curve for distant metastasis free survival for patients whose tumors expressed the combined T47D metagenes (WT or KR, -/+R5020) relative to patient tumors lacking these metagenes. Patient samples include untreated and tamoxifen-treated ER-positive tumors from the Loi et al. dataset [ 29 ]. ( D ) Survival curves as in part C for patients whose tumors expressed the combined T47D metagenes (KR -R5020, or KR +R5020) relative to patient tumors lacking these metagenes. [See also Additional files 4 and 9 ]. ER, estrogen receptor; KR, K388R PR-B mutant; LD, ligand dependent; SUMO, small ubiquitin-like modifier; WT, wild type.
Figure Legend Snippet: The SUMO-deficient PR gene expression signature is associated with HER2-positive human breast tumors and predicts reduced patient survival . ( A ) Normalized gene expression levels (for genes in our LD KR > WT gene signature) are presented for each tumor in the patient cohort [ 64 ], organized by ERBB2 status. ( B ) Gene expression levels were measured by RT-qPCR for CHN2 and RGS2 (both upregulated by SUMO-deficient PR, and members of the LD KR > WT gene signature) and the control gene ACOT6 (equally upregulated by both WT and KR receptors) in BT-474 human breast cancer cells. Cells were pre-treated with MEK kinase inhibitor U0126 prior to progestin or antiprogestin co-treatment. Protein levels were evaluated by western blotting for total PR, PR Ser294 phosphorylation, total ERK1/2, and ERK1/2 phosphorylation. ( C ) Kaplan-Meier survival curve for distant metastasis free survival for patients whose tumors expressed the combined T47D metagenes (WT or KR, -/+R5020) relative to patient tumors lacking these metagenes. Patient samples include untreated and tamoxifen-treated ER-positive tumors from the Loi et al. dataset [ 29 ]. ( D ) Survival curves as in part C for patients whose tumors expressed the combined T47D metagenes (KR -R5020, or KR +R5020) relative to patient tumors lacking these metagenes. [See also Additional files 4 and 9 ]. ER, estrogen receptor; KR, K388R PR-B mutant; LD, ligand dependent; SUMO, small ubiquitin-like modifier; WT, wild type.

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Mutagenesis

Gene expression profiling of T47D cells stably expressing WT or SUMO-deficient PR, treated with or without R5020 for six hours . ( A ) Western blot showing total and phospho-Ser294 PR proteins (total ERK1/2 served as a loading control) in 12 human breast tumors. ( B ) T47D cells stably expressing either wild-type PR-B (WT), SUMO-deficient mutant K388R PR-B (KR), or empty vector (null) controls were treated without or with R5020 prior to western blotting for PR-B. ( C ) Heat map showing normalized expression values for differentially expressed transcripts (fold change > 8.0 in at least one sample, BH adjusted P
Figure Legend Snippet: Gene expression profiling of T47D cells stably expressing WT or SUMO-deficient PR, treated with or without R5020 for six hours . ( A ) Western blot showing total and phospho-Ser294 PR proteins (total ERK1/2 served as a loading control) in 12 human breast tumors. ( B ) T47D cells stably expressing either wild-type PR-B (WT), SUMO-deficient mutant K388R PR-B (KR), or empty vector (null) controls were treated without or with R5020 prior to western blotting for PR-B. ( C ) Heat map showing normalized expression values for differentially expressed transcripts (fold change > 8.0 in at least one sample, BH adjusted P

Techniques Used: Expressing, Stable Transfection, Western Blot, Mutagenesis, Plasmid Preparation

Promoter selectivity is achieved through increased recruitment of SUMO-deficient KR PR, CBP, MLL2 and histone tail modification, H3K4me2, to enhancer loci . ( A ) Schematic showing the MSX2 gene PRE-containing enhancer region located 15,094 bp upstream from the transcriptional start site. ( B ) Relative recruitment of PR to the MSX2 enhancer region was measured by ChIP-qPCR assays in T47D cells expressing constitutive PR null, WT or KR PR after treatment with R5020 for one or four hours. PR recruitment values were normalized as a percentage of input chromatin DNA values. To control for background non-specific antibody binding, immunoprecipitated chromatin contained a mixture from all samples with an IgG antibody. Similar ChIP results were obtained in T47D cells expressing inducible PR (right side). ( C ) The relative recruitment of CBP to the MSX2 enhancer region was measured as described in part B . ( D ) Levels of H3K4 dimethylation at the MSX2 enhancer were measured in the inducible PR expressing cell lines (iWT and iKR). The presence of H3K4me2 was determined at the MSX2 enhancer, up/downstream from the PRE, using overlapping qPCR products that span the region. ( E ) MLL2 recruitment to the MSX2 enhancer region was determined in T47D cells expressing both constitutive PR and inducible PR, as described in part B . ( F ) MAT2A gene expression was measured by RT-qPCR in T47D cells expressing stable WT or SUMO-deficient KR PR. Additionally, PR and MLL2 recruitment was quantified in these cells, as measured by standard ChIP-qPCR assay. Data are represented as mean of n = 3 +/- SD and significance calculated using Student's t-test. [See also Additional files 7 , 5 . KR, K388R PR-B mutant]. CBP, CREB-(cAMP-response element-binding protein)-binding protein; ChIP, chromatin immunoprecipitation; H3K4me2, histone H3 lysine 4 dimethylation; IgG, immunoglobulin G; KR, K388R PR-B mutant; MLL2, mixed lineage leukemia 2; PR, progesterone receptor; PRE, progesterone receptor response element; SD, standard deviation; SUMO, small ubiquitin-like modifier; WT, wild type.
Figure Legend Snippet: Promoter selectivity is achieved through increased recruitment of SUMO-deficient KR PR, CBP, MLL2 and histone tail modification, H3K4me2, to enhancer loci . ( A ) Schematic showing the MSX2 gene PRE-containing enhancer region located 15,094 bp upstream from the transcriptional start site. ( B ) Relative recruitment of PR to the MSX2 enhancer region was measured by ChIP-qPCR assays in T47D cells expressing constitutive PR null, WT or KR PR after treatment with R5020 for one or four hours. PR recruitment values were normalized as a percentage of input chromatin DNA values. To control for background non-specific antibody binding, immunoprecipitated chromatin contained a mixture from all samples with an IgG antibody. Similar ChIP results were obtained in T47D cells expressing inducible PR (right side). ( C ) The relative recruitment of CBP to the MSX2 enhancer region was measured as described in part B . ( D ) Levels of H3K4 dimethylation at the MSX2 enhancer were measured in the inducible PR expressing cell lines (iWT and iKR). The presence of H3K4me2 was determined at the MSX2 enhancer, up/downstream from the PRE, using overlapping qPCR products that span the region. ( E ) MLL2 recruitment to the MSX2 enhancer region was determined in T47D cells expressing both constitutive PR and inducible PR, as described in part B . ( F ) MAT2A gene expression was measured by RT-qPCR in T47D cells expressing stable WT or SUMO-deficient KR PR. Additionally, PR and MLL2 recruitment was quantified in these cells, as measured by standard ChIP-qPCR assay. Data are represented as mean of n = 3 +/- SD and significance calculated using Student's t-test. [See also Additional files 7 , 5 . KR, K388R PR-B mutant]. CBP, CREB-(cAMP-response element-binding protein)-binding protein; ChIP, chromatin immunoprecipitation; H3K4me2, histone H3 lysine 4 dimethylation; IgG, immunoglobulin G; KR, K388R PR-B mutant; MLL2, mixed lineage leukemia 2; PR, progesterone receptor; PRE, progesterone receptor response element; SD, standard deviation; SUMO, small ubiquitin-like modifier; WT, wild type.

Techniques Used: Modification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Binding Assay, Immunoprecipitation, Quantitative RT-PCR, Mutagenesis, Protein Binding, Standard Deviation

10) Product Images from "MMP-9 and CXCL8/IL-8 Are Potential Therapeutic Targets in Epidermolysis Bullosa Simplex"

Article Title: MMP-9 and CXCL8/IL-8 Are Potential Therapeutic Targets in Epidermolysis Bullosa Simplex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0070123

Reduced expression of target genes upon IL-1β depletion. NEB-1 cells and EBDM-1 cells were incubated with 2 µg/ml IL-1β neutralizing antibody for 24 h to deplete IL-1β in the culture medium. Expression of untreated EBDM-1 cells (−) was compared to treated EBDM-1 cells (+) by SQRT-PCR. We picked at least one representative target gene of each of the six functional groups identified in the microarray. All investigated targets showed significant downregulation after IL-1β depletion. No differences in geneexpression were observed in NEB-1 cells −/+ antibody incubation (n = 3). Student’s t -test was performed with p values: * ≤0.05, *** ≤0.005, ΔΔ ≤0.0005.
Figure Legend Snippet: Reduced expression of target genes upon IL-1β depletion. NEB-1 cells and EBDM-1 cells were incubated with 2 µg/ml IL-1β neutralizing antibody for 24 h to deplete IL-1β in the culture medium. Expression of untreated EBDM-1 cells (−) was compared to treated EBDM-1 cells (+) by SQRT-PCR. We picked at least one representative target gene of each of the six functional groups identified in the microarray. All investigated targets showed significant downregulation after IL-1β depletion. No differences in geneexpression were observed in NEB-1 cells −/+ antibody incubation (n = 3). Student’s t -test was performed with p values: * ≤0.05, *** ≤0.005, ΔΔ ≤0.0005.

Techniques Used: Expressing, Incubation, Polymerase Chain Reaction, Functional Assay, Microarray

11) Product Images from "The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts"

Article Title: The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts

Journal: BMC Cancer

doi: 10.1186/1471-2407-13-585

MEFs expressing EWS/ WT1- KTS or EWS/ WT1 + KTS have distinct expression profiles and EWS/ WT1 + KTS expression is associated with up - regulation of canonical Wnt pathway signaling. (A) Heat map of differentially expressed probes identified in primary MEFs expressing EWS/WT1 + KTS, eGFP controls and EWS/WT1-KTS. The comparison is of four independently generated pools of MEFs each infected with EWS/WT1 + KTS, EWS/WT1-KTS or GFP. (B) GSEA plot depicting enrichment of the KIM_WT1_TARGETS_UP gene set in lines expressing EWS/WT1-KTS compared to eGFP controls. (C) Wnt related gene sets enriched in EWS/WT1 + KTS expressing cells (left) and GSEA plot of PID_WNT_Signaling_Pathway geneset (right). (D) Immunocytochemistry of 293 T cells infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS. Cells are shown in the absence of doxycyline (panel on top and also with the presence of doxycycline treatment (thus doxycycline repressed) for 48 hours (panel on bottom). Cells are stained with DAPI for nuclear staining (blue) and β-catenin antibody with secondary anti-goat Alexa Fluor (red). β-catenin translocation from the cellular membrane to the nucleus is a marker of canonical Wnt-pathway activation.
Figure Legend Snippet: MEFs expressing EWS/ WT1- KTS or EWS/ WT1 + KTS have distinct expression profiles and EWS/ WT1 + KTS expression is associated with up - regulation of canonical Wnt pathway signaling. (A) Heat map of differentially expressed probes identified in primary MEFs expressing EWS/WT1 + KTS, eGFP controls and EWS/WT1-KTS. The comparison is of four independently generated pools of MEFs each infected with EWS/WT1 + KTS, EWS/WT1-KTS or GFP. (B) GSEA plot depicting enrichment of the KIM_WT1_TARGETS_UP gene set in lines expressing EWS/WT1-KTS compared to eGFP controls. (C) Wnt related gene sets enriched in EWS/WT1 + KTS expressing cells (left) and GSEA plot of PID_WNT_Signaling_Pathway geneset (right). (D) Immunocytochemistry of 293 T cells infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS. Cells are shown in the absence of doxycyline (panel on top and also with the presence of doxycycline treatment (thus doxycycline repressed) for 48 hours (panel on bottom). Cells are stained with DAPI for nuclear staining (blue) and β-catenin antibody with secondary anti-goat Alexa Fluor (red). β-catenin translocation from the cellular membrane to the nucleus is a marker of canonical Wnt-pathway activation.

Techniques Used: Expressing, Generated, Infection, Immunocytochemistry, Staining, Translocation Assay, Marker, Activation Assay

EWS/WT1-KTS and EWS/WT1 + KTS enhances clonogenicity in p53 +/- or p53 -/- MEFs in reduced serum. (A) Western blot analysis of lysates derived from wild-type, p53 +/- , and p53 -/- background MEFs which were infected with doxycyline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS lentiviral particles, showing expression of EWS/WT1 in the absence of doxycycline. (B) Representative images from one experiment showing MEF colonies of the indicated genotype expressing eGFP, EWS/WT1-KTS or EWS/WT1 + KTS, plated at 1× 10 5 cells per plate and cultured in 1% or 2% fetal calf serum. After 14 days cells were fixed with glutaraldehyde and stained with crystal violet. (C) Mean number of colonies (from three independent experiments) greater than 5 mm of cells cultured in 1% (left panel) or 2% (right panel) serum on day 14. Error bars show ± SEM from three independent experiments. # denotes a p value of
Figure Legend Snippet: EWS/WT1-KTS and EWS/WT1 + KTS enhances clonogenicity in p53 +/- or p53 -/- MEFs in reduced serum. (A) Western blot analysis of lysates derived from wild-type, p53 +/- , and p53 -/- background MEFs which were infected with doxycyline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS lentiviral particles, showing expression of EWS/WT1 in the absence of doxycycline. (B) Representative images from one experiment showing MEF colonies of the indicated genotype expressing eGFP, EWS/WT1-KTS or EWS/WT1 + KTS, plated at 1× 10 5 cells per plate and cultured in 1% or 2% fetal calf serum. After 14 days cells were fixed with glutaraldehyde and stained with crystal violet. (C) Mean number of colonies (from three independent experiments) greater than 5 mm of cells cultured in 1% (left panel) or 2% (right panel) serum on day 14. Error bars show ± SEM from three independent experiments. # denotes a p value of

Techniques Used: Western Blot, Derivative Assay, Infection, Expressing, Cell Culture, Staining

EWS/WT1-KTS and EWS/WT1 + KTS increase anchorage independent growth and confer resistance to daunorubin induced apoptosis and cell cycle arrest following radiation. (A) 1x10 4 p53 wild-type, p53 +/- or p53 -/- MEFs expressing eGFP, EWS/WT1-KTS or EWS/WT1 + KTS were plated in soft agar and the number of colonies greater than 2 mm counted after 14 days. Values are mean ± SEM of three independently generated and infected pools of MEFs for each genotype tested in three independent experiments. P values of less than 0.05 as determined by Student’s t-tests are shown. (B) Viability assay of cells treated with daunorubicin (0.5 μg/ml) for 24 hours. The data show the ratio of WST1 absorbance of treated cells to the same number of untreated cells. Values are mean ± SEM of three independent experiments. P values
Figure Legend Snippet: EWS/WT1-KTS and EWS/WT1 + KTS increase anchorage independent growth and confer resistance to daunorubin induced apoptosis and cell cycle arrest following radiation. (A) 1x10 4 p53 wild-type, p53 +/- or p53 -/- MEFs expressing eGFP, EWS/WT1-KTS or EWS/WT1 + KTS were plated in soft agar and the number of colonies greater than 2 mm counted after 14 days. Values are mean ± SEM of three independently generated and infected pools of MEFs for each genotype tested in three independent experiments. P values of less than 0.05 as determined by Student’s t-tests are shown. (B) Viability assay of cells treated with daunorubicin (0.5 μg/ml) for 24 hours. The data show the ratio of WST1 absorbance of treated cells to the same number of untreated cells. Values are mean ± SEM of three independent experiments. P values

Techniques Used: Expressing, Generated, Infection, Viability Assay

EWS/WT1-KTS and EWS/WT1 + KTS expression co-operates with loss or inactivation of p53 to transform MEFs. (A) Western blots of lysates from SV40 transformed MEFs expressing either eGFP, EWS/WT1-KTS or EWS/WT1 + KTS under the control of a 4-OHT inducible promoter 48 hours after 4-OHT treatment (upper panel) or a doxycycline repressible promoter without doxycycline (lower panel). The anti-WT1 antibody detects a Cterminal-epitope in WT1. Arrow indicates EWS/WT1 to distinguish it from endogenous WT1. A non-specific band of similar size to EWS/WT1 was observed in SV40-transformed, but not untransformed MEFs. (B) qPCR analysis of mRNA expression of eGFP, EWS/WT1-KTS or EWS/WT1 + KTS in MEFs induced by either the 4-OHT inducible or tetracycline repressible expression systems. (C) Fold change in cell number 14 days after MEFs transformed by either SV40 or EIA/RAS were infected with eGFP, EWS/WT1-KTS or EWS/WT1 + KTS using the 4OHT inducible system in SV40 transformed cells, and the doxycycline repressible system in EIA/RAS transformed cells. Cells were plated at equal densities and counted and re-plated at the same dilution every 3–4 days. Data represent the mean ± SEM of three independently generated pools of MEFs tested in three independent experiments. (D) MEFs derived from littermate wildtype (upper panel), p53+/- (middle panel) and p53-/-(lower panel) mice were infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS and then plated at equal density. Cells were counted and re-plated on the indicated days. Values are mean ± SEM of three independently generated and infected pools of MEFs tested over three independent experiments. # denotes a p value of 0.003 and * denotes a p value of 0.004 with Student’s t-test comparing eGFP to EWS/WT1–KTS and eGFP to EWS/WT1 + KTS. Representative images of morphology of MEFs at 22 days are shown.
Figure Legend Snippet: EWS/WT1-KTS and EWS/WT1 + KTS expression co-operates with loss or inactivation of p53 to transform MEFs. (A) Western blots of lysates from SV40 transformed MEFs expressing either eGFP, EWS/WT1-KTS or EWS/WT1 + KTS under the control of a 4-OHT inducible promoter 48 hours after 4-OHT treatment (upper panel) or a doxycycline repressible promoter without doxycycline (lower panel). The anti-WT1 antibody detects a Cterminal-epitope in WT1. Arrow indicates EWS/WT1 to distinguish it from endogenous WT1. A non-specific band of similar size to EWS/WT1 was observed in SV40-transformed, but not untransformed MEFs. (B) qPCR analysis of mRNA expression of eGFP, EWS/WT1-KTS or EWS/WT1 + KTS in MEFs induced by either the 4-OHT inducible or tetracycline repressible expression systems. (C) Fold change in cell number 14 days after MEFs transformed by either SV40 or EIA/RAS were infected with eGFP, EWS/WT1-KTS or EWS/WT1 + KTS using the 4OHT inducible system in SV40 transformed cells, and the doxycycline repressible system in EIA/RAS transformed cells. Cells were plated at equal densities and counted and re-plated at the same dilution every 3–4 days. Data represent the mean ± SEM of three independently generated pools of MEFs tested in three independent experiments. (D) MEFs derived from littermate wildtype (upper panel), p53+/- (middle panel) and p53-/-(lower panel) mice were infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS and then plated at equal density. Cells were counted and re-plated on the indicated days. Values are mean ± SEM of three independently generated and infected pools of MEFs tested over three independent experiments. # denotes a p value of 0.003 and * denotes a p value of 0.004 with Student’s t-test comparing eGFP to EWS/WT1–KTS and eGFP to EWS/WT1 + KTS. Representative images of morphology of MEFs at 22 days are shown.

Techniques Used: Expressing, Western Blot, Transformation Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Infection, Generated, Derivative Assay, Mouse Assay

MEFs expressing EWS/ WT1- KTS or EWS/ WT1 + KTS have distinct expression profiles and EWS/ WT1 + KTS expression is associated with up - regulation of canonical Wnt pathway signaling. (A) Heat map of differentially expressed probes identified in primary MEFs expressing EWS/WT1 + KTS, eGFP controls and EWS/WT1-KTS. The comparison is of four independently generated pools of MEFs each infected with EWS/WT1 + KTS, EWS/WT1-KTS or GFP. (B) GSEA plot depicting enrichment of the KIM_WT1_TARGETS_UP gene set in lines expressing EWS/WT1-KTS compared to eGFP controls. (C) Wnt related gene sets enriched in EWS/WT1 + KTS expressing cells (left) and GSEA plot of PID_WNT_Signaling_Pathway geneset (right). (D) Immunocytochemistry of 293 T cells infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS. Cells are shown in the absence of doxycyline (panel on top and also with the presence of doxycycline treatment (thus doxycycline repressed) for 48 hours (panel on bottom). Cells are stained with DAPI for nuclear staining (blue) and β-catenin antibody with secondary anti-goat Alexa Fluor (red). β-catenin translocation from the cellular membrane to the nucleus is a marker of canonical Wnt-pathway activation.
Figure Legend Snippet: MEFs expressing EWS/ WT1- KTS or EWS/ WT1 + KTS have distinct expression profiles and EWS/ WT1 + KTS expression is associated with up - regulation of canonical Wnt pathway signaling. (A) Heat map of differentially expressed probes identified in primary MEFs expressing EWS/WT1 + KTS, eGFP controls and EWS/WT1-KTS. The comparison is of four independently generated pools of MEFs each infected with EWS/WT1 + KTS, EWS/WT1-KTS or GFP. (B) GSEA plot depicting enrichment of the KIM_WT1_TARGETS_UP gene set in lines expressing EWS/WT1-KTS compared to eGFP controls. (C) Wnt related gene sets enriched in EWS/WT1 + KTS expressing cells (left) and GSEA plot of PID_WNT_Signaling_Pathway geneset (right). (D) Immunocytochemistry of 293 T cells infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS. Cells are shown in the absence of doxycyline (panel on top and also with the presence of doxycycline treatment (thus doxycycline repressed) for 48 hours (panel on bottom). Cells are stained with DAPI for nuclear staining (blue) and β-catenin antibody with secondary anti-goat Alexa Fluor (red). β-catenin translocation from the cellular membrane to the nucleus is a marker of canonical Wnt-pathway activation.

Techniques Used: Expressing, Generated, Infection, Immunocytochemistry, Staining, Translocation Assay, Marker, Activation Assay

12) Product Images from "MiR-133b Regulates the Expression of the Actin Protein TAGLN2 during Oocyte Growth and Maturation: A Potential Target for Infertility Therapy"

Article Title: MiR-133b Regulates the Expression of the Actin Protein TAGLN2 during Oocyte Growth and Maturation: A Potential Target for Infertility Therapy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0100751

miR-133b targets TAGLN2 in 293T cells. A) Vertical axis is the ratio of Renilla to firefly luciferase and the horizontal axis is the experiment groups: 1. siCHECK-NP; 2. siCHECK-3UTR+microRNA-NC; 3. siCHECK-3UTR+25 nM microRNA-133b; 4. siCHECK-3UTR+50 nM microRNA-133b; 5. siCHECK-3UTR+100 nM microRA-133b; 6. siCHECK-3UTR-m+100 nM microRNA-133b. B) Protein expression of TAGLN2 in 293T cells after being treated with miR-133b. C) mRNA expression of TAGLN2 in 293T cells after being treated with miR-133b. Each sample was tested in triplicate and the data were presented in mean ± SD. The experiments were repeated twice and similar results were obtained. ** P
Figure Legend Snippet: miR-133b targets TAGLN2 in 293T cells. A) Vertical axis is the ratio of Renilla to firefly luciferase and the horizontal axis is the experiment groups: 1. siCHECK-NP; 2. siCHECK-3UTR+microRNA-NC; 3. siCHECK-3UTR+25 nM microRNA-133b; 4. siCHECK-3UTR+50 nM microRNA-133b; 5. siCHECK-3UTR+100 nM microRA-133b; 6. siCHECK-3UTR-m+100 nM microRNA-133b. B) Protein expression of TAGLN2 in 293T cells after being treated with miR-133b. C) mRNA expression of TAGLN2 in 293T cells after being treated with miR-133b. Each sample was tested in triplicate and the data were presented in mean ± SD. The experiments were repeated twice and similar results were obtained. ** P

Techniques Used: Luciferase, Expressing

13) Product Images from "Two Functionally Distinct Isoforms of TL1A (TNFSF15) Generated by Differential Ectodomain Shedding"

Article Title: Two Functionally Distinct Isoforms of TL1A (TNFSF15) Generated by Differential Ectodomain Shedding

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

doi: 10.1093/gerona/glq129

Upregulation of tumor necrosis factor–like cytokine 1A (TL1A) messenger RNA (mRNA) in replicative senescent circulating endothelial progenitor cells (CEP) as well as in elderly donors. (A) CEP of a young donor (Y2 = 35 years) were serially passaged until they reached senescence. mRNA was prepared and subjected to cDNA microarray analysis. Microarray results were normalized, and the regulation for TNFSF15 probe set (TL1A) is shown. (B) In addition, CEP of young (Y1 = 30 years) and old donors (O1 = 70 years and O2 = 79 years) were isolated and analyzed by cDNA microarray. (C) The mRNA expression observed by cDNA microarray analysis was confirmed by real-time-quantitative-PCR (RTq-PCR) using primers specific for the detection of TL1A. Results are shown as fold change compared with “young” CEP regarding replicative age or donor age, respectively. (D) CEP derived from three young donors (Y3, Y4, and Y5) were transduced with either control lentivirus or three lentiviral knockdown vectors targeting TL1A (pLKO1, pLKO99). RTq-PCR showing the lentiviral knockdown of TL1A in CEP was performed 8 and 10 days after transduction at the indicated passage numbers. Results are shown as fold change compared with control. (E) Quantification of senescence-associated β-galactosidase (SA-β-gal)–positive cells after knockdown of TL1A. CEP of young donors (Y3, Y4 and Y5) were fixed and stained for SA-β-gal 7 or 9 days after transduction. Phase contrast microscopic pictures were taken at 400× magnification. The percentage of SA-β-gal–positive cells was calculated by counting about 1,500 cells per experiment. (F) CEP were transduced with lentiviral vectors expressing green fluorescent protein (GFP) or TL1A, and cell extracts were subjected to Western blot using GFP or TL1A-specific antibodies, respectively. Wild-type CEP were used as a control. (G) Six days after transduction, cells were fixed and stained for SA-β-gal. Pictures are representative of at least three experiments.
Figure Legend Snippet: Upregulation of tumor necrosis factor–like cytokine 1A (TL1A) messenger RNA (mRNA) in replicative senescent circulating endothelial progenitor cells (CEP) as well as in elderly donors. (A) CEP of a young donor (Y2 = 35 years) were serially passaged until they reached senescence. mRNA was prepared and subjected to cDNA microarray analysis. Microarray results were normalized, and the regulation for TNFSF15 probe set (TL1A) is shown. (B) In addition, CEP of young (Y1 = 30 years) and old donors (O1 = 70 years and O2 = 79 years) were isolated and analyzed by cDNA microarray. (C) The mRNA expression observed by cDNA microarray analysis was confirmed by real-time-quantitative-PCR (RTq-PCR) using primers specific for the detection of TL1A. Results are shown as fold change compared with “young” CEP regarding replicative age or donor age, respectively. (D) CEP derived from three young donors (Y3, Y4, and Y5) were transduced with either control lentivirus or three lentiviral knockdown vectors targeting TL1A (pLKO1, pLKO99). RTq-PCR showing the lentiviral knockdown of TL1A in CEP was performed 8 and 10 days after transduction at the indicated passage numbers. Results are shown as fold change compared with control. (E) Quantification of senescence-associated β-galactosidase (SA-β-gal)–positive cells after knockdown of TL1A. CEP of young donors (Y3, Y4 and Y5) were fixed and stained for SA-β-gal 7 or 9 days after transduction. Phase contrast microscopic pictures were taken at 400× magnification. The percentage of SA-β-gal–positive cells was calculated by counting about 1,500 cells per experiment. (F) CEP were transduced with lentiviral vectors expressing green fluorescent protein (GFP) or TL1A, and cell extracts were subjected to Western blot using GFP or TL1A-specific antibodies, respectively. Wild-type CEP were used as a control. (G) Six days after transduction, cells were fixed and stained for SA-β-gal. Pictures are representative of at least three experiments.

Techniques Used: Microarray, Isolation, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Derivative Assay, Transduction, Staining, Western Blot

14) Product Images from "An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice"

Article Title: An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice

Journal: Nature genetics

doi: 10.1038/ng.369

Ocm , Slc26a5 , Pitpnm1 , Ptpr1 and Gfi1 expression in diminuendo a , Quantitative real-time PCR on cDNA generated from normalised RNA from the organs of Corti of 4 day old littermates. Ocm , Slc26a5 , Ptprq and Gfi1 are downregulated in heterozygotes and homozygotes. Error bars represent standard deviation. Quantities normalised to Hprt1 levels; Ngfr is expressed in support cells adjacent to hair cells 30 and was used to assess the quantity of sensory material. Ngfr : Wildtype, n=12, mean=1.01±0.12 (s.d.); heterozygote, n=12, mean=0.89±0.35 (s.d.); homozygote, n=12, mean=0.99±0.17 (s.d.) Ocm : Wildtype, n=9, mean=1.01±0.15 (s.d.); heterozygote, n=9, mean=0.07±0.04 (s.d.); homozygote, n=9, mean=0.003±0.001 (s.d.) Slc26a5 : Wildtype, n=9, mean=1.01±0.14 (s.d.); heterozygote, n=9, mean=0.22±0.11 (s.d.); homozygote, n=9, mean=0.02±0.01 (s.d.) Gfi1 : Wildtype, n=9, mean=1.01±0.12 (s.d.); heterozygote, n=9, mean=0.88±0.12 (s.d.); homozygote, n=9, mean=0.66±0.14 (s.d.) Ptprq : Wildtype, n=9, mean=1.01±0.15 (s.d.); heterozygote, n=9, mean=0.62±0.18 (s.d.); homozygote, n=9, mean=0.56±0.20 (s.d.) Pitpnm1 : Wildtype n=8, mean=1.00±0.09 (s.d.); heterozygote, n=9, mean=0.80±0.29 (s.d.); homozygote, n=9, mean=0.78±0.27 (s.d.). Three animals were used for each genotype and DNA from each was run in triplicate. T-tests: Ngfr heterozygote p=0.25 (Welch's t-test), homozygote p=0.75 (Student's t-test); Ocm heterozygote p=1.51×10 −8 (Welch's t-test), homozygote p=3.46×10 −8 (Welch's t-test); Slc26a5 heterozygote p=7.73×10 −10 (Student's t-test), homozygote p=3.37×10 −8 (Welch's t-test); Gfi1 heterozygote p=0.038 (Student's t-test), homozygote p=3.39×10 −5 (Student's t-test); Ptprq heterozygote p=1.37×10 −4 (Student's t-test), homozygote p=6.46×10 −5 (Student's t-test); Pitpnm1 heterozygote p=0.084 (Welch's t-test), homozygote p=0.35 (Student's t-test); α=0.05. b-k , location of oncomodulin ( b, c ), prestin ( d, h ), Pitpnm1 ( e , i ), Ptprq ( f , j ) and Gfi1 ( g , k ) in 5-day old wildtype ( b, d-g ) and homozygote ( c, h-k ) littermates. Scale bars = 10μm.
Figure Legend Snippet: Ocm , Slc26a5 , Pitpnm1 , Ptpr1 and Gfi1 expression in diminuendo a , Quantitative real-time PCR on cDNA generated from normalised RNA from the organs of Corti of 4 day old littermates. Ocm , Slc26a5 , Ptprq and Gfi1 are downregulated in heterozygotes and homozygotes. Error bars represent standard deviation. Quantities normalised to Hprt1 levels; Ngfr is expressed in support cells adjacent to hair cells 30 and was used to assess the quantity of sensory material. Ngfr : Wildtype, n=12, mean=1.01±0.12 (s.d.); heterozygote, n=12, mean=0.89±0.35 (s.d.); homozygote, n=12, mean=0.99±0.17 (s.d.) Ocm : Wildtype, n=9, mean=1.01±0.15 (s.d.); heterozygote, n=9, mean=0.07±0.04 (s.d.); homozygote, n=9, mean=0.003±0.001 (s.d.) Slc26a5 : Wildtype, n=9, mean=1.01±0.14 (s.d.); heterozygote, n=9, mean=0.22±0.11 (s.d.); homozygote, n=9, mean=0.02±0.01 (s.d.) Gfi1 : Wildtype, n=9, mean=1.01±0.12 (s.d.); heterozygote, n=9, mean=0.88±0.12 (s.d.); homozygote, n=9, mean=0.66±0.14 (s.d.) Ptprq : Wildtype, n=9, mean=1.01±0.15 (s.d.); heterozygote, n=9, mean=0.62±0.18 (s.d.); homozygote, n=9, mean=0.56±0.20 (s.d.) Pitpnm1 : Wildtype n=8, mean=1.00±0.09 (s.d.); heterozygote, n=9, mean=0.80±0.29 (s.d.); homozygote, n=9, mean=0.78±0.27 (s.d.). Three animals were used for each genotype and DNA from each was run in triplicate. T-tests: Ngfr heterozygote p=0.25 (Welch's t-test), homozygote p=0.75 (Student's t-test); Ocm heterozygote p=1.51×10 −8 (Welch's t-test), homozygote p=3.46×10 −8 (Welch's t-test); Slc26a5 heterozygote p=7.73×10 −10 (Student's t-test), homozygote p=3.37×10 −8 (Welch's t-test); Gfi1 heterozygote p=0.038 (Student's t-test), homozygote p=3.39×10 −5 (Student's t-test); Ptprq heterozygote p=1.37×10 −4 (Student's t-test), homozygote p=6.46×10 −5 (Student's t-test); Pitpnm1 heterozygote p=0.084 (Welch's t-test), homozygote p=0.35 (Student's t-test); α=0.05. b-k , location of oncomodulin ( b, c ), prestin ( d, h ), Pitpnm1 ( e , i ), Ptprq ( f , j ) and Gfi1 ( g , k ) in 5-day old wildtype ( b, d-g ) and homozygote ( c, h-k ) littermates. Scale bars = 10μm.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Generated, Standard Deviation

15) Product Images from "Relative importance of redox buffers GSH and NAD(P)H in age-related neurodegeneration and Alzheimer disease-like mouse neurons"

Article Title: Relative importance of redox buffers GSH and NAD(P)H in age-related neurodegeneration and Alzheimer disease-like mouse neurons

Journal: Aging Cell

doi: 10.1111/acel.12216

Brain NADPH concentration and NADPH/NADP redox state decline with age and correlate with decline in brain expression of NAMPT and NNT gene expression. By HPLC analysis of cortical/hippocampal tissue homogenates from 4-, 11- and 21-month non-Tg (dashed line) and 3xTg-AD (solid line), A) NADPH concentration (nmol/mg) declines with age (ANOVA F(2,24) = 10.4, P = 0.001). B) Calculated NADPH/NADP redox state (mV) using the Nernst equation indicates a large oxidative shift in both non-Tg and 3xTg-AD brain with age (ANOVA F(2,24) = 15.7, P = 0.001). qRT PCR on non-Tg (gray filled circle, dashed line) and 3xTg-AD (black filled circle, solid line) brains indicate an age- and AD-related loss in gene expression of metabolic enzymes C) NAMPT (ANOVA, age F(3,33) = 21.8, P
Figure Legend Snippet: Brain NADPH concentration and NADPH/NADP redox state decline with age and correlate with decline in brain expression of NAMPT and NNT gene expression. By HPLC analysis of cortical/hippocampal tissue homogenates from 4-, 11- and 21-month non-Tg (dashed line) and 3xTg-AD (solid line), A) NADPH concentration (nmol/mg) declines with age (ANOVA F(2,24) = 10.4, P = 0.001). B) Calculated NADPH/NADP redox state (mV) using the Nernst equation indicates a large oxidative shift in both non-Tg and 3xTg-AD brain with age (ANOVA F(2,24) = 15.7, P = 0.001). qRT PCR on non-Tg (gray filled circle, dashed line) and 3xTg-AD (black filled circle, solid line) brains indicate an age- and AD-related loss in gene expression of metabolic enzymes C) NAMPT (ANOVA, age F(3,33) = 21.8, P

Techniques Used: Concentration Assay, Expressing, High Performance Liquid Chromatography, Quantitative RT-PCR

16) Product Images from "The Pathogen Recognition Receptor NOD2 Regulates Human FOXP3+ T Cell Survival"

Article Title: The Pathogen Recognition Receptor NOD2 Regulates Human FOXP3+ T Cell Survival

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.0901479

Nod2 mRNA is expressed in human Treg cells. A , RNA extracted from freshly isolated CD4 + CD25 ++ Treg cells, CD4 − PBMCs, and 293T cells is probed for Nod2, CD11b, and actin. Expression of Nod2 mRNA within Treg cells and CD4 − PBMCs (CD4 −
Figure Legend Snippet: Nod2 mRNA is expressed in human Treg cells. A , RNA extracted from freshly isolated CD4 + CD25 ++ Treg cells, CD4 − PBMCs, and 293T cells is probed for Nod2, CD11b, and actin. Expression of Nod2 mRNA within Treg cells and CD4 − PBMCs (CD4 −

Techniques Used: Isolation, Expressing

17) Product Images from "CRISPR Screen in Regulatory T Cells Reveals Ubiquitination Modulators of Foxp3"

Article Title: CRISPR Screen in Regulatory T Cells Reveals Ubiquitination Modulators of Foxp3

Journal: bioRxiv

doi: 10.1101/2020.02.26.966911

Usp22 is required to maintain Treg suppressive function and Foxp3 stability in vivo. a) Representative flow cytometry analysis of the Treg population (gated on CD4 + cells) from the spleen of Treg-specific Usp22 WT and KO mice. The Foxp3 hi CD25 hi subpopulation is highlighted with a red gate. b) Histogram of Foxp3 expression from the spleen of Treg-specific Usp22 WT and KO mice from panel a. c) Statistical analysis of Foxp3 MFI from thymus (Thy), peripheral lymph nodes (pLN) and spleen (Spl). *P
Figure Legend Snippet: Usp22 is required to maintain Treg suppressive function and Foxp3 stability in vivo. a) Representative flow cytometry analysis of the Treg population (gated on CD4 + cells) from the spleen of Treg-specific Usp22 WT and KO mice. The Foxp3 hi CD25 hi subpopulation is highlighted with a red gate. b) Histogram of Foxp3 expression from the spleen of Treg-specific Usp22 WT and KO mice from panel a. c) Statistical analysis of Foxp3 MFI from thymus (Thy), peripheral lymph nodes (pLN) and spleen (Spl). *P

Techniques Used: In Vivo, Flow Cytometry, Mouse Assay, Expressing

Usp22 Regulates Foxp3 through Transcriptional Mechanisms. a) Statistical analysis of CD4 + YFP + Treg frequencies, corresponding to Figure 3c . b) Schematic of Foxp3 locus depicting PCR products used for ChIP-qPCR. c) ChIP-qPCR data analysis for H2A-ub for PCR across the transcriptional start site (TSS) and across the CNS1 enhancer region. Data are normalized to the input.
Figure Legend Snippet: Usp22 Regulates Foxp3 through Transcriptional Mechanisms. a) Statistical analysis of CD4 + YFP + Treg frequencies, corresponding to Figure 3c . b) Schematic of Foxp3 locus depicting PCR products used for ChIP-qPCR. c) ChIP-qPCR data analysis for H2A-ub for PCR across the transcriptional start site (TSS) and across the CNS1 enhancer region. Data are normalized to the input.

Techniques Used: Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Usp22 Regulates Foxp3 Transcription by Reciprocal Ubiquitin Modulators. a) Representative flow cytometry analysis of the YFP + Treg population (gated on CD4 + cells) from the spleen and lymph nodes of Treg-specific Usp22 WT and KO mice. b) Histogram of YFP expression from the spleen and lymph nodes of Treg-specific Usp22 WT and KO mice from panel a. c) Statistical analysis of YFP MFI from thymus (Thy), peripheral lymph nodes (pLN) and spleen (Spl), n=3-4. d) qPCR analysis mRNA level of Foxp3 in sorted YFP+ cells of spleen from Usp22 WT and KO mice. ns indicates no significant difference, *P
Figure Legend Snippet: Usp22 Regulates Foxp3 Transcription by Reciprocal Ubiquitin Modulators. a) Representative flow cytometry analysis of the YFP + Treg population (gated on CD4 + cells) from the spleen and lymph nodes of Treg-specific Usp22 WT and KO mice. b) Histogram of YFP expression from the spleen and lymph nodes of Treg-specific Usp22 WT and KO mice from panel a. c) Statistical analysis of YFP MFI from thymus (Thy), peripheral lymph nodes (pLN) and spleen (Spl), n=3-4. d) qPCR analysis mRNA level of Foxp3 in sorted YFP+ cells of spleen from Usp22 WT and KO mice. ns indicates no significant difference, *P

Techniques Used: Flow Cytometry, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

Treg-Specific Ablation of Usp22 Results in Spontaneous Autoimmune Symptoms and Enhances Anti-Tumor Immunity a) Body weight differences between Usp22 WT and KO littermate mice. b) Hematoxylin-and-eosin (H E) staining of kidney, lung, colon and liver sections from 7-month-old WT and KO mice. Original magnification, x100. c) Weight of Rag -/- recipient mice (n=4-5 per group) over time after adoptive transfer of CD4 + CD25 - CD44 lo CD62 hi (CD45.1 + ) naïve T cells sorted from SJL mice alone or together with CD4 + YFP + (CD45.2 + ) Treg cells from 9-week-old Usp22 WT or KO mice, presented relative to weight at day 0, set as 1. d) H E staining of colon tissues from the Rag -/- recipient mice shown in panel c, 7 weeks post transfer. Original magnification at 100x (fold). e) Clinical severity of EAE in Usp22 WT and KO mice (n=4) was monitored until day 20 post immunization with MOG peptide. f) EG7 lymphoma tumor growth is inhibited in Usp22 KO mice. Mice (n=4 or 5 per group) were subcutaneously inoculated with 106 EG7 cells. The tumor volume was measured every 2 or 3 days by scaling along 3 orthogonal axes (x, y, and z) and calculated as (xyz)/2. g) Representative flow cytometry analysis of the expression of CD44 and CD62L in both CD4+ and CD8+ T cells of spleen from EG7 tumor-bearing mice. h) The percentage of effector memory-like activated T cells (CD44 hi CD62L lo ) from EG7 tumor-bearing mice summarized. i) Statistical analysis of tumor-infiltrating lymphocyte (TIL) percentages from EG7-bearing Usp22 WT or KO mice after tumor inoculation. j) Statistical analysis of tumor-infiltrating Treg percentages from EG7-bearing Usp22 WT or KO mice after tumor inoculation. k) Foxp3 MFI of the CD4+Foxp3+ EG7 tumor-infiltrating Treg population summarized, (n=5-7). Data are mean ±SEM and are representative of three independent experiments. ns indicates no significant difference, *P
Figure Legend Snippet: Treg-Specific Ablation of Usp22 Results in Spontaneous Autoimmune Symptoms and Enhances Anti-Tumor Immunity a) Body weight differences between Usp22 WT and KO littermate mice. b) Hematoxylin-and-eosin (H E) staining of kidney, lung, colon and liver sections from 7-month-old WT and KO mice. Original magnification, x100. c) Weight of Rag -/- recipient mice (n=4-5 per group) over time after adoptive transfer of CD4 + CD25 - CD44 lo CD62 hi (CD45.1 + ) naïve T cells sorted from SJL mice alone or together with CD4 + YFP + (CD45.2 + ) Treg cells from 9-week-old Usp22 WT or KO mice, presented relative to weight at day 0, set as 1. d) H E staining of colon tissues from the Rag -/- recipient mice shown in panel c, 7 weeks post transfer. Original magnification at 100x (fold). e) Clinical severity of EAE in Usp22 WT and KO mice (n=4) was monitored until day 20 post immunization with MOG peptide. f) EG7 lymphoma tumor growth is inhibited in Usp22 KO mice. Mice (n=4 or 5 per group) were subcutaneously inoculated with 106 EG7 cells. The tumor volume was measured every 2 or 3 days by scaling along 3 orthogonal axes (x, y, and z) and calculated as (xyz)/2. g) Representative flow cytometry analysis of the expression of CD44 and CD62L in both CD4+ and CD8+ T cells of spleen from EG7 tumor-bearing mice. h) The percentage of effector memory-like activated T cells (CD44 hi CD62L lo ) from EG7 tumor-bearing mice summarized. i) Statistical analysis of tumor-infiltrating lymphocyte (TIL) percentages from EG7-bearing Usp22 WT or KO mice after tumor inoculation. j) Statistical analysis of tumor-infiltrating Treg percentages from EG7-bearing Usp22 WT or KO mice after tumor inoculation. k) Foxp3 MFI of the CD4+Foxp3+ EG7 tumor-infiltrating Treg population summarized, (n=5-7). Data are mean ±SEM and are representative of three independent experiments. ns indicates no significant difference, *P

Techniques Used: Mouse Assay, Staining, Adoptive Transfer Assay, Flow Cytometry, Expressing

Design and Validation of Treg-specific Usp22 Knockout Mice. a) Diagram of the murine Usp22 locus. Targeting vector contains IRES-lacZ and a neo cassette inserted into exon 2. b) Genotyping by PCR showed a 600-bp band for the wild-type allele and a 400-bp band for mutant allele, simultaneously in the homozygous floxed (f/f) mice. c) Western blot analysis of Usp22 in CD4 + CD25 - conventional T cells (Tconv) and CD4 + CD25 + Treg cells isolated from WT and KO mice. Tubulin was used as a loading control. d) Statistical analysis of CD4 + CD25 + Foxp3 + Treg frequencies, n=4, corresponding to Figure 2c . e) iTreg differentiations from WT and KO naïve CD4 + T cells with titration of TGF-β.
Figure Legend Snippet: Design and Validation of Treg-specific Usp22 Knockout Mice. a) Diagram of the murine Usp22 locus. Targeting vector contains IRES-lacZ and a neo cassette inserted into exon 2. b) Genotyping by PCR showed a 600-bp band for the wild-type allele and a 400-bp band for mutant allele, simultaneously in the homozygous floxed (f/f) mice. c) Western blot analysis of Usp22 in CD4 + CD25 - conventional T cells (Tconv) and CD4 + CD25 + Treg cells isolated from WT and KO mice. Tubulin was used as a loading control. d) Statistical analysis of CD4 + CD25 + Foxp3 + Treg frequencies, n=4, corresponding to Figure 2c . e) iTreg differentiations from WT and KO naïve CD4 + T cells with titration of TGF-β.

Techniques Used: Knock-Out, Mouse Assay, Plasmid Preparation, Polymerase Chain Reaction, Mutagenesis, Western Blot, Isolation, Titration

Usp22 Acts as Deubiquitinase to Control Post-Translational Foxp3 Expression. a) Endogenous interaction of Usp22 and Foxp3 in murine iTreg cells from WT mice. Rabbit anti-Usp22 antibody was used to perform the immunoprecipitation and mouse anti-Foxp3 antibody was used to detect the bound Foxp3. Normal rabbit IgG was used as control. b) Ubiquitination assay of Foxp3 with WT or mutant Usp22, where the conserved cysteine (C) residue in the C19 peptidase domain was replaced by an alanine (A) residue. c) Splenocytes isolated from Usp22 WT or KO mice were treated with 200 μg/ml cycloheximide (CHX) for the indicated time course. Inset numbers for each histogram indicate the MFI of Foxp3 (black=WT, blue=KO). d) Foxp3 MFI from the CD4 + CD25 + Foxp3 + Treg population (n=3). Data are mean ±SEM and are representative of three independent experiments. *P
Figure Legend Snippet: Usp22 Acts as Deubiquitinase to Control Post-Translational Foxp3 Expression. a) Endogenous interaction of Usp22 and Foxp3 in murine iTreg cells from WT mice. Rabbit anti-Usp22 antibody was used to perform the immunoprecipitation and mouse anti-Foxp3 antibody was used to detect the bound Foxp3. Normal rabbit IgG was used as control. b) Ubiquitination assay of Foxp3 with WT or mutant Usp22, where the conserved cysteine (C) residue in the C19 peptidase domain was replaced by an alanine (A) residue. c) Splenocytes isolated from Usp22 WT or KO mice were treated with 200 μg/ml cycloheximide (CHX) for the indicated time course. Inset numbers for each histogram indicate the MFI of Foxp3 (black=WT, blue=KO). d) Foxp3 MFI from the CD4 + CD25 + Foxp3 + Treg population (n=3). Data are mean ±SEM and are representative of three independent experiments. *P

Techniques Used: Expressing, Mouse Assay, Immunoprecipitation, Ubiquitin Assay, Mutagenesis, Isolation

Design and Quality Control of Targeted Pooled CRISPR Screen in Primary Mouse Tregs a) Design strategy for selection of genes for unbiased targeted library of 493 targets, including 490 nuclear factors and 3 control targets (NT, GFP, and RFP). Genes were selected based on gene ontology (GO) annotation and then sub-selected based on highest expression across any CD4 T cell subset for a total of 2,000 sgRNAs. b) Diagram of MSCV expression vector with Thy1.1 reporter used for retroviral transduction of the sgRNA library. c) Detailed timeline schematic of the 12-day targeted screen pipeline. Arrows indicate when the cells were split and media was replenished. d) Retroviral transduction efficiency of the targeted library in primary mouse Tregs shown by Thy1.1 surface expression measured by flow cytometry. The infection was scaled to achieve a high efficiency multiplicity of infection. e) Foxp3 expression from screen input, output and control cells measured by flow cytometry. Top: Foxp3 expression from input Foxp3 + purified Tregs as measured by GFP expression on Day 0. Middle: Foxp3 expression as measured by endogenous intracellular staining from control Tregs (not transduced with library) on Day 12. Bottom: Foxp3 expression as measured by endogenous intracellular staining from screen Tregs (transduced with library) on Day 12. f) Targeted screen (2,000 guides) shows that sgRNAs targeting Foxp3 and Usp22 were enriched in Foxp3 low cells (blue). Non-targeting control (NT Ctrl) sgRNAs were evenly distributed across the cell populations (black). g) Distribution of read counts after next generation sequencing of sgRNAs of sorted cell populations, Foxp3 high and Foxp3 low .
Figure Legend Snippet: Design and Quality Control of Targeted Pooled CRISPR Screen in Primary Mouse Tregs a) Design strategy for selection of genes for unbiased targeted library of 493 targets, including 490 nuclear factors and 3 control targets (NT, GFP, and RFP). Genes were selected based on gene ontology (GO) annotation and then sub-selected based on highest expression across any CD4 T cell subset for a total of 2,000 sgRNAs. b) Diagram of MSCV expression vector with Thy1.1 reporter used for retroviral transduction of the sgRNA library. c) Detailed timeline schematic of the 12-day targeted screen pipeline. Arrows indicate when the cells were split and media was replenished. d) Retroviral transduction efficiency of the targeted library in primary mouse Tregs shown by Thy1.1 surface expression measured by flow cytometry. The infection was scaled to achieve a high efficiency multiplicity of infection. e) Foxp3 expression from screen input, output and control cells measured by flow cytometry. Top: Foxp3 expression from input Foxp3 + purified Tregs as measured by GFP expression on Day 0. Middle: Foxp3 expression as measured by endogenous intracellular staining from control Tregs (not transduced with library) on Day 12. Bottom: Foxp3 expression as measured by endogenous intracellular staining from screen Tregs (transduced with library) on Day 12. f) Targeted screen (2,000 guides) shows that sgRNAs targeting Foxp3 and Usp22 were enriched in Foxp3 low cells (blue). Non-targeting control (NT Ctrl) sgRNAs were evenly distributed across the cell populations (black). g) Distribution of read counts after next generation sequencing of sgRNAs of sorted cell populations, Foxp3 high and Foxp3 low .

Techniques Used: CRISPR, Selection, Expressing, Plasmid Preparation, Transduction, Flow Cytometry, Infection, Purification, Staining, Next-Generation Sequencing

18) Product Images from "Short chain fatty acid butyrate promotes virus infection by repressing interferon stimulated genes"

Article Title: Short chain fatty acid butyrate promotes virus infection by repressing interferon stimulated genes

Journal: bioRxiv

doi: 10.1101/2020.02.04.934919

Butyrate differentially regulates baseline and IFN-induced expression of ISGs. A549 cells were mock treated or pretreated with butyrate (2.5 mM) for 1 h prior to stimulation with IFNβ or vehicle control for 8 h in the continued presence or absence of butyrate. a-f) RNA was extracted and subjected to RNA-seq analysis. Three biological replicates for each condition were analyzed by RNA-seq. a) Volcano plot of differentially expressed genes comparing mock treated and butyrate treated cells. b) Gene ontology term analysis of the top 5 biological processes associated with genes upregulated by butyrate. c) Venn diagram comparing baseline butyrate-regulated genes (4-fold or greater increase or decrease in response to butyrate treatment) to genes classified as ISGs (genes showing a 4-fold or greater increase in expression stimulated by IFNβ). d) Pie chart representing number of ISGs categorized based on the effect of butyrate on their mRNA levels. Downregulated genes were classified as a 4-fold or greater decrease in expression stimulated by IFNβ in the presence of butyrate. Upregulated genes were classified as a 4-fold or greater increase in expression stimulated by IFNβ in the presence of butyrate. Not strongly affected indicates all other ISGs. e) Heat map of all ISGs under the indicated conditions compared to mock treated cells (without butyrate or IFNβ treatment). Data represented are the mean of 3 biological replicates per condition. f) Magnification of specific regions in the heat map shown in e to visualize representative example ISGs belonging to each of the 3 ISG categories based on the effect of butyrate on their induction. g) Independent experimental samples were prepared as in a-f for validation of RNA-seq results via qRT-PCR. Fold change is expressed relative to mock treated cells (without butyrate or IFNβ treatment). Gene expression was normalized to GAPDH for each sample. Open circles represent triplicate samples in a single experiment. Bars represent average values and error bars represent standard deviation. Horizontal lines indicate statistical comparisons of interest. *** P
Figure Legend Snippet: Butyrate differentially regulates baseline and IFN-induced expression of ISGs. A549 cells were mock treated or pretreated with butyrate (2.5 mM) for 1 h prior to stimulation with IFNβ or vehicle control for 8 h in the continued presence or absence of butyrate. a-f) RNA was extracted and subjected to RNA-seq analysis. Three biological replicates for each condition were analyzed by RNA-seq. a) Volcano plot of differentially expressed genes comparing mock treated and butyrate treated cells. b) Gene ontology term analysis of the top 5 biological processes associated with genes upregulated by butyrate. c) Venn diagram comparing baseline butyrate-regulated genes (4-fold or greater increase or decrease in response to butyrate treatment) to genes classified as ISGs (genes showing a 4-fold or greater increase in expression stimulated by IFNβ). d) Pie chart representing number of ISGs categorized based on the effect of butyrate on their mRNA levels. Downregulated genes were classified as a 4-fold or greater decrease in expression stimulated by IFNβ in the presence of butyrate. Upregulated genes were classified as a 4-fold or greater increase in expression stimulated by IFNβ in the presence of butyrate. Not strongly affected indicates all other ISGs. e) Heat map of all ISGs under the indicated conditions compared to mock treated cells (without butyrate or IFNβ treatment). Data represented are the mean of 3 biological replicates per condition. f) Magnification of specific regions in the heat map shown in e to visualize representative example ISGs belonging to each of the 3 ISG categories based on the effect of butyrate on their induction. g) Independent experimental samples were prepared as in a-f for validation of RNA-seq results via qRT-PCR. Fold change is expressed relative to mock treated cells (without butyrate or IFNβ treatment). Gene expression was normalized to GAPDH for each sample. Open circles represent triplicate samples in a single experiment. Bars represent average values and error bars represent standard deviation. Horizontal lines indicate statistical comparisons of interest. *** P

Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Standard Deviation

19) Product Images from "Loss of function variants in PCYT1A causing spondylometaphyseal dysplasia with cone/rod dystrophy have broad consequences on lipid metabolism, chondrocyte differentiation, and lipid droplet formation"

Article Title: Loss of function variants in PCYT1A causing spondylometaphyseal dysplasia with cone/rod dystrophy have broad consequences on lipid metabolism, chondrocyte differentiation, and lipid droplet formation

Journal: bioRxiv

doi: 10.1101/2019.12.19.882191

Lipid droplet phenotype and rescue experiments. Lipid droplet (LD) formation was induced by oleic acid loading (1 mM, 24 hr) and visualized with BODIPY staining. LD size and number were quantified using “analyze particle” command in ImageJ (v1.47). Note heterogeneity in LDs. A and C) Neither LD size (p=0.43) nor number (p=0.87) differed between control fibroblasts and SMD-CRD patient fibroblasts. B and D) PCYT1A -null HEK293 cells have fewer and larger LDs than control HEK293 cells. E) LD sizes and numbers are rescued by transfection of PCYT1A -null HEK293T cells with PCYT1A WT, p.Ala99Val or p.Tyr240His cDNAs but not corresponding empty vector controls. n=200 HEK293 PCYT1A null cells from 4 experimental trials. Error bars represent SD. We used Welch Two Sample t-test for panels C and D, and Tukey’s Honest Significant Difference test for panel E. Data in panel E is represented as box and whisker plot for which the box represents the range of the first to third quartiles, line represents the median, and whiskers extend to minimum and maximum data points. *p
Figure Legend Snippet: Lipid droplet phenotype and rescue experiments. Lipid droplet (LD) formation was induced by oleic acid loading (1 mM, 24 hr) and visualized with BODIPY staining. LD size and number were quantified using “analyze particle” command in ImageJ (v1.47). Note heterogeneity in LDs. A and C) Neither LD size (p=0.43) nor number (p=0.87) differed between control fibroblasts and SMD-CRD patient fibroblasts. B and D) PCYT1A -null HEK293 cells have fewer and larger LDs than control HEK293 cells. E) LD sizes and numbers are rescued by transfection of PCYT1A -null HEK293T cells with PCYT1A WT, p.Ala99Val or p.Tyr240His cDNAs but not corresponding empty vector controls. n=200 HEK293 PCYT1A null cells from 4 experimental trials. Error bars represent SD. We used Welch Two Sample t-test for panels C and D, and Tukey’s Honest Significant Difference test for panel E. Data in panel E is represented as box and whisker plot for which the box represents the range of the first to third quartiles, line represents the median, and whiskers extend to minimum and maximum data points. *p

Techniques Used: Staining, Transfection, Plasmid Preparation, Whisker Assay

PCYT1A -null HEK293 and ATDC5 cells have normal proliferation rates. A) Wild-type and PCYT1A -null HEK293 cells were grown in either normal serum or delipidated serum up to 144 hours and counted every 24 hours. Error bars represent SD. B) Undifferentiated wild-type or Pcyt1a -null ATDC5 cells were grown for 5 days in normal serum and counted every 24 hours using absorbance-based cell counting. Error bars represent SD of three replicates.
Figure Legend Snippet: PCYT1A -null HEK293 and ATDC5 cells have normal proliferation rates. A) Wild-type and PCYT1A -null HEK293 cells were grown in either normal serum or delipidated serum up to 144 hours and counted every 24 hours. Error bars represent SD. B) Undifferentiated wild-type or Pcyt1a -null ATDC5 cells were grown for 5 days in normal serum and counted every 24 hours using absorbance-based cell counting. Error bars represent SD of three replicates.

Techniques Used: Cell Counting

Lipidomics reveal decreases in LPCs and in PC:PE ratio in SMD-CRD patient fibroblasts and PCYT1A -null HEK293 cells. A) Data represent mean values of relative abundance of lysophosphatidylcholine (LPC) species of various acyl chain lengths and compositions relative to internal standards in SMD-CRD patient fibroblasts and PCYT1A -null HEK293 cells as compared to wild-type controls. Error bars represent SD of three replicates. p
Figure Legend Snippet: Lipidomics reveal decreases in LPCs and in PC:PE ratio in SMD-CRD patient fibroblasts and PCYT1A -null HEK293 cells. A) Data represent mean values of relative abundance of lysophosphatidylcholine (LPC) species of various acyl chain lengths and compositions relative to internal standards in SMD-CRD patient fibroblasts and PCYT1A -null HEK293 cells as compared to wild-type controls. Error bars represent SD of three replicates. p

Techniques Used:

Involvement of Pcyt1a in ATDC5 chondrocyte differentiation. A) Western blot analysis of ATDC5 cell lysates shows that CCTα steady-state levels remain stable over the course of 21 days of chondrocyte differentiation. B) Rates of chondrocyte differentiation in control and Pcyt1a -null ATDC5 cells were measured by absorbance of Alcian blue in SDS-lysed cell extracts. n=3 biological replicates for each time point.
Figure Legend Snippet: Involvement of Pcyt1a in ATDC5 chondrocyte differentiation. A) Western blot analysis of ATDC5 cell lysates shows that CCTα steady-state levels remain stable over the course of 21 days of chondrocyte differentiation. B) Rates of chondrocyte differentiation in control and Pcyt1a -null ATDC5 cells were measured by absorbance of Alcian blue in SDS-lysed cell extracts. n=3 biological replicates for each time point.

Techniques Used: Western Blot

20) Product Images from "Multiplexed imaging of nucleome architectures in single cells of mammalian tissue"

Article Title: Multiplexed imaging of nucleome architectures in single cells of mammalian tissue

Journal: bioRxiv

doi: 10.1101/2019.12.20.885277

Mapping multiscale chromatin folding, RNA profiles, and other cellular and nuclear components in single cells of mammalian tissue. ( A ) Schematic illustration of the biological features measured by Multiplexed Imaging of Nucleome Architectures (MINA). We imaged cell boundaries, nuclei, nucleoli, 137 different RNA species, the center 100-kb regions of 50 TADs in chromosome 19 (Chr19), and 19 consecutive 5-kb loci upstream of gene Scd2 in E14.5 mouse fetal liver tissue sections. ( B ) A simplified scheme of the chromatin tracing approach. All genomic regions were first labeled with a library of primary probes in a primary hybridization (Hyb0), and then sequentially visualized with dye-labeled secondary probes in a series of secondary hybridization (Hyb1, 2, 3…), imaging, and bleaching steps. ( C ) (Left panels) Individual images of three TADs and sum image of all TADs in a copy of Chr19. Images are max projections along the z direction of the 3D image stacks. Fitted TAD center positions are projected onto the sum image. (Right panel) 3D positions of TADs plotted as pseudo-colored spheres connected with a smooth curve. ( D ) (Left panels) Individual and sum images of the probed 5-kb loci. Images are max projections along the z direction of the 3D image stacks. Fitted locus positions are projected onto the sum image. (Right panel) 3D positions of the loci plotted as spheres connected with a smooth curve. ( E ) (Left panel) Image of cell nuclei (blue) and nucleoli (yellow) labeling. (Right panel) Computationally abstracted nuclear periphery (blue) and nucleoli (yellow) regions. ( F ) A simplified scheme of the RNA profiling approach. A library of primary probes was first hybridized to the RNA molecules, which encoded each RNA species with a unique16-bit barcode. Then the barcode on each RNA molecule was decoded with a series of secondary hybridization, imaging and bleaching steps. ( G ) (i-iii) Images of RNA molecules in three rounds of secondary hybridization. Images are from a single z position in the 3D image stacks. (iv) All identified RNA molecules in a field of view pseudo-colored based on their gene identities. The yellow boxed region is the same region shown in i-iii. ( H ) (Top left) Image of cell boundary labeling. (Bottom right) Computationally abstracted cell boundaries. ( I ) Mean spatial distance matrix of the 50 TADs, with each element of the matrix showing the mean spatial distance between a pair of TADs. ( J ) Inverse Hi-C contact frequency versus mean spatial distance for each pair of TADs. ( K ) Total RNA copy numbers from imaging versus FPKM values from bulk RNA sequencing for each probed RNA species.
Figure Legend Snippet: Mapping multiscale chromatin folding, RNA profiles, and other cellular and nuclear components in single cells of mammalian tissue. ( A ) Schematic illustration of the biological features measured by Multiplexed Imaging of Nucleome Architectures (MINA). We imaged cell boundaries, nuclei, nucleoli, 137 different RNA species, the center 100-kb regions of 50 TADs in chromosome 19 (Chr19), and 19 consecutive 5-kb loci upstream of gene Scd2 in E14.5 mouse fetal liver tissue sections. ( B ) A simplified scheme of the chromatin tracing approach. All genomic regions were first labeled with a library of primary probes in a primary hybridization (Hyb0), and then sequentially visualized with dye-labeled secondary probes in a series of secondary hybridization (Hyb1, 2, 3…), imaging, and bleaching steps. ( C ) (Left panels) Individual images of three TADs and sum image of all TADs in a copy of Chr19. Images are max projections along the z direction of the 3D image stacks. Fitted TAD center positions are projected onto the sum image. (Right panel) 3D positions of TADs plotted as pseudo-colored spheres connected with a smooth curve. ( D ) (Left panels) Individual and sum images of the probed 5-kb loci. Images are max projections along the z direction of the 3D image stacks. Fitted locus positions are projected onto the sum image. (Right panel) 3D positions of the loci plotted as spheres connected with a smooth curve. ( E ) (Left panel) Image of cell nuclei (blue) and nucleoli (yellow) labeling. (Right panel) Computationally abstracted nuclear periphery (blue) and nucleoli (yellow) regions. ( F ) A simplified scheme of the RNA profiling approach. A library of primary probes was first hybridized to the RNA molecules, which encoded each RNA species with a unique16-bit barcode. Then the barcode on each RNA molecule was decoded with a series of secondary hybridization, imaging and bleaching steps. ( G ) (i-iii) Images of RNA molecules in three rounds of secondary hybridization. Images are from a single z position in the 3D image stacks. (iv) All identified RNA molecules in a field of view pseudo-colored based on their gene identities. The yellow boxed region is the same region shown in i-iii. ( H ) (Top left) Image of cell boundary labeling. (Bottom right) Computationally abstracted cell boundaries. ( I ) Mean spatial distance matrix of the 50 TADs, with each element of the matrix showing the mean spatial distance between a pair of TADs. ( J ) Inverse Hi-C contact frequency versus mean spatial distance for each pair of TADs. ( K ) Total RNA copy numbers from imaging versus FPKM values from bulk RNA sequencing for each probed RNA species.

Techniques Used: Imaging, Labeling, Hybridization, Hi-C, RNA Sequencing Assay

21) Product Images from "Gain-of-function mutation in Gli3 causes ventricular septal defects"

Article Title: Gain-of-function mutation in Gli3 causes ventricular septal defects

Journal: bioRxiv

doi: 10.1101/2020.02.10.942144

Reduced proliferation rate in ciliated regions of Gli3 Δ699/Δ699 embryonic hearts. (A, B) Immunofluorescence on mouse embryonic heart sections obtained from WT (n=12), Gli3 Δ699/Δ699 (n=8) and Gli3 XtJ/XtJ (n=12) embryos at E12.5. Proliferating cells are marked by pH3 (A) or KI67 (B). Cell nuclei are marked by DAPI. Scale bars represent a length of 100 µm in the overview and 15 µm in the insets. LV, left ventricle; RV, right ventricle.
Figure Legend Snippet: Reduced proliferation rate in ciliated regions of Gli3 Δ699/Δ699 embryonic hearts. (A, B) Immunofluorescence on mouse embryonic heart sections obtained from WT (n=12), Gli3 Δ699/Δ699 (n=8) and Gli3 XtJ/XtJ (n=12) embryos at E12.5. Proliferating cells are marked by pH3 (A) or KI67 (B). Cell nuclei are marked by DAPI. Scale bars represent a length of 100 µm in the overview and 15 µm in the insets. LV, left ventricle; RV, right ventricle.

Techniques Used: Immunofluorescence

PDGFRα signalling activators rescue proliferation rate in Gli3 Δ699/Δ699 MEFs. (A-C) Immunofluorescence on and ciliary length quantifications in MEFs obtained from WT and Gli3 Δ699/Δ699 (n = 4, respectively) embryos at E12.5. (A) The ciliary axoneme is stained in green by acetylated α-tubulin, the basal body is stained in blue by γ-tubulin. The amount of PDGFRα is significantly reduced in cilia of Gli3 Δ699/Δ699 MEFs. The scale bar represents a length of 0.5 µm. (B) Ciliary length measurement. (C) Proliferating cells are marked by pH3. Cell nuclei are marked by DAPI.
Figure Legend Snippet: PDGFRα signalling activators rescue proliferation rate in Gli3 Δ699/Δ699 MEFs. (A-C) Immunofluorescence on and ciliary length quantifications in MEFs obtained from WT and Gli3 Δ699/Δ699 (n = 4, respectively) embryos at E12.5. (A) The ciliary axoneme is stained in green by acetylated α-tubulin, the basal body is stained in blue by γ-tubulin. The amount of PDGFRα is significantly reduced in cilia of Gli3 Δ699/Δ699 MEFs. The scale bar represents a length of 0.5 µm. (B) Ciliary length measurement. (C) Proliferating cells are marked by pH3. Cell nuclei are marked by DAPI.

Techniques Used: Immunofluorescence, Staining

Gli3 Δ699/Δ699 mutant embryos display VSDs and a decreased HH signalling. (A) HE staining of heart sections from WT, Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ embryos at E14.5. Asterisk indicates VSD. The scale bar represents a length of 500 µm. (B, C) Quantification of Gli1 (B) and Ptch1 (C) expression via qRT-PCR analysis. Lysates were obtained from WT, Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ (n = 3, respectively) mouse embryonic hearts at E12.5. (D) Western blot analysis of GLI2 with lysates obtained from Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ (n = 3, respectively) mouse embryonic hearts at E12.5. Actin serves as loading control.
Figure Legend Snippet: Gli3 Δ699/Δ699 mutant embryos display VSDs and a decreased HH signalling. (A) HE staining of heart sections from WT, Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ embryos at E14.5. Asterisk indicates VSD. The scale bar represents a length of 500 µm. (B, C) Quantification of Gli1 (B) and Ptch1 (C) expression via qRT-PCR analysis. Lysates were obtained from WT, Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ (n = 3, respectively) mouse embryonic hearts at E12.5. (D) Western blot analysis of GLI2 with lysates obtained from Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ (n = 3, respectively) mouse embryonic hearts at E12.5. Actin serves as loading control.

Techniques Used: Mutagenesis, Staining, Expressing, Quantitative RT-PCR, Western Blot

Reduced PDGFRα signalling and ciliary length in Gli3 Δ699/Δ699 hearts. (A) Immunofluorescence on heart sections obtained from WT (n=12), Gli3 Δ699/Δ699 (n=8) and Gli3 XtJ/XtJ (n=12) embryos at E12.5. The ciliary axoneme is stained in green by acetylated α-tubulin, the basal body is stained in blue by γ-tubulin. The scale bar represents a length of 0.5 µm. (B) Ciliary length measurement on mouse embryonic heart sections obtained from WT, Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ (n = 4, respectively) embryos at E12.5.
Figure Legend Snippet: Reduced PDGFRα signalling and ciliary length in Gli3 Δ699/Δ699 hearts. (A) Immunofluorescence on heart sections obtained from WT (n=12), Gli3 Δ699/Δ699 (n=8) and Gli3 XtJ/XtJ (n=12) embryos at E12.5. The ciliary axoneme is stained in green by acetylated α-tubulin, the basal body is stained in blue by γ-tubulin. The scale bar represents a length of 0.5 µm. (B) Ciliary length measurement on mouse embryonic heart sections obtained from WT, Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ (n = 4, respectively) embryos at E12.5.

Techniques Used: Immunofluorescence, Staining

22) Product Images from "Linear ubiquitin assembly complex regulates lung epithelial–driven responses during influenza infection"

Article Title: Linear ubiquitin assembly complex regulates lung epithelial–driven responses during influenza infection

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI128368

HOIL-1L is upregulated during IAV infection through a type I IFN receptor signaling axis. ( A and B ) AT2 cells were isolated from WT mice at 0, 3, 5, and 7 d.p.i. ( A ) HOIL-1L mRNA ( n = 5). ( B ) Representative HOIL-1L immunoblot and its quantification ( n = 4). ( C ) Representative native PAGE immunoblot of HOIL-1L and HOIP expression in AT2 cells infected in vitro with WSN ( n = 3). ( D ) HOIL-1L mRNA expression in AT2 cells from WT mice 0 and 3 d.p.i. sorted based on expression of the viral protein HA ( n = 8). ( E – I ) Representative HOIL-1L immunoblots and quantification. ( E ) A549 cells treated for 0, 8, 12, and 16 hours with conditioned medium (CM; “16*” indicates boiled CM) ( n = 4). ( F ) A549 cells treated with recombinant IFN-α ( n = 4). ( G ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with CM ( n = 3). ( H ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with WSN ( n = 3). ( I ) AT2 cells isolated from WT and CCR2 –/– mice treated in vitro with WSN ( n = 4). ( J ) Quantitative reverse transcriptase PCR quantification of HOIL-1L promoter after ChIP of IRF1 in A549 cells ( n = 4). ( K ) Representative HOIL-1L immunoblot and quantification in siControl- or siIRF1-transfected A549 cells treated with CM ( n = 4). ( L ) Proposed type I IFN pathway leading to HOIL-1L upregulation. Means ± SD overlaid with individual data points representing replicates are depicted; ** P
Figure Legend Snippet: HOIL-1L is upregulated during IAV infection through a type I IFN receptor signaling axis. ( A and B ) AT2 cells were isolated from WT mice at 0, 3, 5, and 7 d.p.i. ( A ) HOIL-1L mRNA ( n = 5). ( B ) Representative HOIL-1L immunoblot and its quantification ( n = 4). ( C ) Representative native PAGE immunoblot of HOIL-1L and HOIP expression in AT2 cells infected in vitro with WSN ( n = 3). ( D ) HOIL-1L mRNA expression in AT2 cells from WT mice 0 and 3 d.p.i. sorted based on expression of the viral protein HA ( n = 8). ( E – I ) Representative HOIL-1L immunoblots and quantification. ( E ) A549 cells treated for 0, 8, 12, and 16 hours with conditioned medium (CM; “16*” indicates boiled CM) ( n = 4). ( F ) A549 cells treated with recombinant IFN-α ( n = 4). ( G ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with CM ( n = 3). ( H ) AT2 cells isolated from WT and IFNAR1 –/– mice treated in vitro with WSN ( n = 3). ( I ) AT2 cells isolated from WT and CCR2 –/– mice treated in vitro with WSN ( n = 4). ( J ) Quantitative reverse transcriptase PCR quantification of HOIL-1L promoter after ChIP of IRF1 in A549 cells ( n = 4). ( K ) Representative HOIL-1L immunoblot and quantification in siControl- or siIRF1-transfected A549 cells treated with CM ( n = 4). ( L ) Proposed type I IFN pathway leading to HOIL-1L upregulation. Means ± SD overlaid with individual data points representing replicates are depicted; ** P

Techniques Used: Infection, Isolation, Mouse Assay, Clear Native PAGE, Expressing, In Vitro, Western Blot, Recombinant, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Transfection

23) Product Images from "STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis"

Article Title: STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0113318

CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Amplification, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.
Figure Legend Snippet: Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.

Techniques Used: Amplification, Isolation, Genome Wide, Expressing

IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Functional Assay, Activity Assay, Amplification, Isolation, Quantitative RT-PCR, Expressing, Migration

Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation

STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p
Figure Legend Snippet: STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p

Techniques Used: Isolation, Quantitative RT-PCR

24) Product Images from "Exendin-4 enhances expression of Neurod1 and Glut2 in insulin-producing cells derived from mouse embryonic stem cells"

Article Title: Exendin-4 enhances expression of Neurod1 and Glut2 in insulin-producing cells derived from mouse embryonic stem cells

Journal: Archives of Medical Science : AMS

doi: 10.5114/aoms.2016.57596

Morphology of RI ES cells, insulin-producing cells derived from different differentiation protocols. A – Morphology of R1 ES cells. B – Alkaline phosphatase (AP) staining of R1 ES cells. C – In vitro formation of simple EBs on day 6 of stage 1. D–F – Insulin-producing cells derived from different differentiation protocols. D – low dosage group, E – high dosage group, F – control group. Scale bars = 25 µm
Figure Legend Snippet: Morphology of RI ES cells, insulin-producing cells derived from different differentiation protocols. A – Morphology of R1 ES cells. B – Alkaline phosphatase (AP) staining of R1 ES cells. C – In vitro formation of simple EBs on day 6 of stage 1. D–F – Insulin-producing cells derived from different differentiation protocols. D – low dosage group, E – high dosage group, F – control group. Scale bars = 25 µm

Techniques Used: Derivative Assay, Staining, In Vitro

Immunostaining of insulin and C-peptide. Day 31 R1 ES cell-derived insulin-producing cells were immunostained with antibodies against insulin ( A ) and C-peptide ( B ). A – Positive signals for insulin were easily observed in groups C, L and H. B – C-peptide positive staining was observed only in groups H and L. Nuclei were stained with DAPI (4’,6-diamidine-2’-phenylindole dihydrochloride). Scale bars = 25 µm
Figure Legend Snippet: Immunostaining of insulin and C-peptide. Day 31 R1 ES cell-derived insulin-producing cells were immunostained with antibodies against insulin ( A ) and C-peptide ( B ). A – Positive signals for insulin were easily observed in groups C, L and H. B – C-peptide positive staining was observed only in groups H and L. Nuclei were stained with DAPI (4’,6-diamidine-2’-phenylindole dihydrochloride). Scale bars = 25 µm

Techniques Used: Immunostaining, Derivative Assay, Staining

25) Product Images from "Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming"

Article Title: Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming

Journal: PLoS ONE

doi: 10.1371/journal.pone.0131288

Ocular epithelial differentiation from different cell-of-origin iPSCs lines. (A) To compare the pluripotency nature of different types of iPSCs, OECiPSCs and OSCiPSCs were subjected to sphere culture for 7 days then monolayer culture for further 3 days. Cells were harvested for testing endodermal, mesodermal and ectodermal markers by real-time PCR. Expression level of pluripotency markers Pou5f1, Sox2 and Dnmt3a were dramatically decreased up to 1000 times in both differences in OECiPSCs and OSCiPSCs. The OSCiPSCs demonstrated a preference for endodermal differentiation while OECiPSCs show higher potential in ectodermal differ nation. iPSCs vs OECiPSCs vs OSCiPSCs: Pou5f1 (0.00±0;-3.56±0.06;-2.98±0.07); Sox2 (0.00±0;-2.83±0.04;-3.25±0.03); Dnmt3a (0.00±0;-2.42±0.03;-3.01±0.04); Sox17 (0.00±0; 1.08±0.04; 1.48±0.03); Foxa2 (0.00±0; 1.35±0.04; 2.12±0.03); AFP (0.00±0; 1.23±0.23; 1.41±0.47); T (0.00±0; 2.21±0.04; 1.41±0.03); Kit (0.00±0; 2.17±0.0; 2.62±0.03); Nestin (0.00±0; 2.65±0.20; 1.56±0.22); Pax6 (0.00±0; 2.75±0.04; 2.23±0.11); (SEM N = 3;* P
Figure Legend Snippet: Ocular epithelial differentiation from different cell-of-origin iPSCs lines. (A) To compare the pluripotency nature of different types of iPSCs, OECiPSCs and OSCiPSCs were subjected to sphere culture for 7 days then monolayer culture for further 3 days. Cells were harvested for testing endodermal, mesodermal and ectodermal markers by real-time PCR. Expression level of pluripotency markers Pou5f1, Sox2 and Dnmt3a were dramatically decreased up to 1000 times in both differences in OECiPSCs and OSCiPSCs. The OSCiPSCs demonstrated a preference for endodermal differentiation while OECiPSCs show higher potential in ectodermal differ nation. iPSCs vs OECiPSCs vs OSCiPSCs: Pou5f1 (0.00±0;-3.56±0.06;-2.98±0.07); Sox2 (0.00±0;-2.83±0.04;-3.25±0.03); Dnmt3a (0.00±0;-2.42±0.03;-3.01±0.04); Sox17 (0.00±0; 1.08±0.04; 1.48±0.03); Foxa2 (0.00±0; 1.35±0.04; 2.12±0.03); AFP (0.00±0; 1.23±0.23; 1.41±0.47); T (0.00±0; 2.21±0.04; 1.41±0.03); Kit (0.00±0; 2.17±0.0; 2.62±0.03); Nestin (0.00±0; 2.65±0.20; 1.56±0.22); Pax6 (0.00±0; 2.75±0.04; 2.23±0.11); (SEM N = 3;* P

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

Endogenous expression of OCT4A and SOX2 pluripotency reprogramming factors in OECs derived from conjunctival tissues. (A) DAB-based immunohistochemistry staining displayed the expression of OCT4A (brown, arrows) in human ocular sections. OCT4A expressed in the epithelium layer but not in stromal layers (OEL, ocular epithelial layer; OSL, ocular stromal layer). (B) Immunofluorescence staining demonstrated OCT4A expression in OECs but not in OSCs. (C) (i) Reverse transcription polymerase chain reaction (RT-PCR) results in expression of pluripotency genes (from top to bottom Panels): DPPA4 , TERT , NANOG , GDF3 , REX1 , ESG1 , OCT4A , SOX2 , KLF4 and C-MYC and Beta- actin . Human ESCs and water were included as positive and negative controls respectively. (ii) Expression of OCT4 isoforms OCT4A and OCT4B, endogenous OCT 4 and viral OCT4 detecting using 5’ and 3’ UTR sequences tracking with RT-PCR. (From left to right) Primary lines (IMR90, OEC1, OEC2 and OSCs) and their corresponding iPSCs. (D) Western Blotting for OCT4A and SOX2 protein levels in OECs, OSCs and iPSCs. Lane 1: OECiPSCs, Lane 2: OSCs, Lane 3 4: OEC1 2 respectively, Lane 5: mouse OECs (E) Expression levels of (i) OCT4A and (ii) SOX2 were represented in intensity ratios respectively.
Figure Legend Snippet: Endogenous expression of OCT4A and SOX2 pluripotency reprogramming factors in OECs derived from conjunctival tissues. (A) DAB-based immunohistochemistry staining displayed the expression of OCT4A (brown, arrows) in human ocular sections. OCT4A expressed in the epithelium layer but not in stromal layers (OEL, ocular epithelial layer; OSL, ocular stromal layer). (B) Immunofluorescence staining demonstrated OCT4A expression in OECs but not in OSCs. (C) (i) Reverse transcription polymerase chain reaction (RT-PCR) results in expression of pluripotency genes (from top to bottom Panels): DPPA4 , TERT , NANOG , GDF3 , REX1 , ESG1 , OCT4A , SOX2 , KLF4 and C-MYC and Beta- actin . Human ESCs and water were included as positive and negative controls respectively. (ii) Expression of OCT4 isoforms OCT4A and OCT4B, endogenous OCT 4 and viral OCT4 detecting using 5’ and 3’ UTR sequences tracking with RT-PCR. (From left to right) Primary lines (IMR90, OEC1, OEC2 and OSCs) and their corresponding iPSCs. (D) Western Blotting for OCT4A and SOX2 protein levels in OECs, OSCs and iPSCs. Lane 1: OECiPSCs, Lane 2: OSCs, Lane 3 4: OEC1 2 respectively, Lane 5: mouse OECs (E) Expression levels of (i) OCT4A and (ii) SOX2 were represented in intensity ratios respectively.

Techniques Used: Expressing, Derivative Assay, Immunohistochemistry, Staining, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot

26) Product Images from "Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210"

Article Title: Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210

Journal: Parasites & Vectors

doi: 10.1186/s13071-015-0974-3

Global analysis of differentially expressed miRNAs in L. m. -infected BMDM, bioinformatical prediction of miRNA interactions with LISA, and infection rates of L. m. -infected BMDM after transfection with mmu-miR-101c or mmu-miR-129-5p mimics as well as mmu-miR-155-5p or mmu-miR-210-5p inhibitors. Methods: ( a , b , d ) BMDM from BALB/c mice were infected with L. m. promastigotes for 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. Affymetrix® chips were hybridized with RNA samples from 2 independent experiments and analyzed densitometrically. Putative interactions between differentially expressed miRNAs and LISA members were predicted bioinformatically and represented in MONA-of-LISA. c Additionally, L. m. -infected BMDM were transfected with miRNA mimics or inhibitors 20 h p.i.. L. m. -infected control BMDM were transfected with a negative control of miRNA mimics or inhibitors. The infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a , b ) Differentially expressed miRNAs were detected in L. m. -infected BMDM 24 h p.i.. The results were presented in MA plots. Large dots represent probe sets, which were significantly and differentially expressed (FDR
Figure Legend Snippet: Global analysis of differentially expressed miRNAs in L. m. -infected BMDM, bioinformatical prediction of miRNA interactions with LISA, and infection rates of L. m. -infected BMDM after transfection with mmu-miR-101c or mmu-miR-129-5p mimics as well as mmu-miR-155-5p or mmu-miR-210-5p inhibitors. Methods: ( a , b , d ) BMDM from BALB/c mice were infected with L. m. promastigotes for 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. Affymetrix® chips were hybridized with RNA samples from 2 independent experiments and analyzed densitometrically. Putative interactions between differentially expressed miRNAs and LISA members were predicted bioinformatically and represented in MONA-of-LISA. c Additionally, L. m. -infected BMDM were transfected with miRNA mimics or inhibitors 20 h p.i.. L. m. -infected control BMDM were transfected with a negative control of miRNA mimics or inhibitors. The infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a , b ) Differentially expressed miRNAs were detected in L. m. -infected BMDM 24 h p.i.. The results were presented in MA plots. Large dots represent probe sets, which were significantly and differentially expressed (FDR

Techniques Used: Infection, Transfection, Mouse Assay, Incubation, Negative Control

BNIP3 and CTSE transcriptomic and western blot analyses with RNAs and protein extracts from L. m. -infected and HBSS-starved BMDM as well as determination of the infection rates of L. m. -infected BMDM after BNIP3 and CTSE downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 h or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. The Proteins were harvested and subjected to western blot analysis with specific antibodies against BNIP3 and CTSE. Western blots from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. Affymetrix® chips were hybridized with RNA samples from 2 independent experiments and analyzed densitometrically. c Additionally, L. m. -infected BMDM were transfected with specific siRNAs 20 h p.i. to downregulate the expression of BNIP3 and CTSE. L. m. -infected control BMDM were transfected with negative control siRNA. Infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a ) BNIP3 and CTSE were significantly overexpressed in L. m. -infected BMDM 24 h p.i. compared to uninfected control BMDM. b Densitometric analyses of western blot experiments confirmed this overexpression 24 h p.i. and showed that CTSE was also overexpressed in L. m. -infected BMDM 1 h p.i.. At the mRNA level, Bnip3 was overexpressed in L. m. -infected BMDM 1 and 24 h p.i. and Ctse was downregulated in L. m. -infected BMDM 24 h p.i.. c A significant increase in the infection rates was detected in L. m. -infected BMDM after downregulation of protein expression of BNIP3 or CTSE compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01,*** p ≤ 0.001
Figure Legend Snippet: BNIP3 and CTSE transcriptomic and western blot analyses with RNAs and protein extracts from L. m. -infected and HBSS-starved BMDM as well as determination of the infection rates of L. m. -infected BMDM after BNIP3 and CTSE downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 h or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. The Proteins were harvested and subjected to western blot analysis with specific antibodies against BNIP3 and CTSE. Western blots from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. Affymetrix® chips were hybridized with RNA samples from 2 independent experiments and analyzed densitometrically. c Additionally, L. m. -infected BMDM were transfected with specific siRNAs 20 h p.i. to downregulate the expression of BNIP3 and CTSE. L. m. -infected control BMDM were transfected with negative control siRNA. Infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a ) BNIP3 and CTSE were significantly overexpressed in L. m. -infected BMDM 24 h p.i. compared to uninfected control BMDM. b Densitometric analyses of western blot experiments confirmed this overexpression 24 h p.i. and showed that CTSE was also overexpressed in L. m. -infected BMDM 1 h p.i.. At the mRNA level, Bnip3 was overexpressed in L. m. -infected BMDM 1 and 24 h p.i. and Ctse was downregulated in L. m. -infected BMDM 24 h p.i.. c A significant increase in the infection rates was detected in L. m. -infected BMDM after downregulation of protein expression of BNIP3 or CTSE compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01,*** p ≤ 0.001

Techniques Used: Western Blot, Infection, Mouse Assay, Incubation, Transfection, Expressing, Negative Control, Over Expression, Fluorescence

MTOR and RPS6 transcriptomic and western blot analyses with RNAs and protein extracts from L. m. -infected and HBSS-starved BMDM, and determination of infection rates of L. m. -infected BMDM after MTOR downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. Proteins were harvested and subjected to western blot analyses with specific antibodies against MTOR, p-MTOR, RPS6, and p-RPS6. Western blots with proteins from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. The Affymetrix® chips were hybridized with RNA samples from 2 independent experiments were analyzed densitometrically. c Additionally, BMDM were transfected with specific siRNA 4 h prior to infection to downregulate the expression of MTOR, and the cells were finally infected with L. m. promastigotes. L. m. -infected controls were transfected with negative control siRNA. The infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a ) A significant hyperphosphorylation was observed for MTOR and RPS6 in samples from L. m. -infected BMDM 1 h p.i. compared to uninfected control BMDM. b Results of densitometric analyses of western blot experiments and Affymetrix® chip analyses showed that MTOR and RPS6 expressions were not regulated at the mRNA or the protein level. However, MTOR and RPS6 were significantly hyperphosphorylated in L. m. -infected BMDM 1 h p.i.. c A significant decrease in the infection rate was detected in L. m. -infected BMDM after downregulation of the protein expression of MTOR compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01
Figure Legend Snippet: MTOR and RPS6 transcriptomic and western blot analyses with RNAs and protein extracts from L. m. -infected and HBSS-starved BMDM, and determination of infection rates of L. m. -infected BMDM after MTOR downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. Proteins were harvested and subjected to western blot analyses with specific antibodies against MTOR, p-MTOR, RPS6, and p-RPS6. Western blots with proteins from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. The Affymetrix® chips were hybridized with RNA samples from 2 independent experiments were analyzed densitometrically. c Additionally, BMDM were transfected with specific siRNA 4 h prior to infection to downregulate the expression of MTOR, and the cells were finally infected with L. m. promastigotes. L. m. -infected controls were transfected with negative control siRNA. The infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a ) A significant hyperphosphorylation was observed for MTOR and RPS6 in samples from L. m. -infected BMDM 1 h p.i. compared to uninfected control BMDM. b Results of densitometric analyses of western blot experiments and Affymetrix® chip analyses showed that MTOR and RPS6 expressions were not regulated at the mRNA or the protein level. However, MTOR and RPS6 were significantly hyperphosphorylated in L. m. -infected BMDM 1 h p.i.. c A significant decrease in the infection rate was detected in L. m. -infected BMDM after downregulation of the protein expression of MTOR compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01

Techniques Used: Western Blot, Infection, Mouse Assay, Incubation, Transfection, Expressing, Negative Control, Chromatin Immunoprecipitation, Fluorescence

Schematic summary of results. a 0 h p.i.: macrophages were infected with L. m. promastigotes. b 10 min p.i.: promastigotes attached to macrophages and were phagocytosed by the cells. c 1 h p.i. (early infection phase): promastigotes differentiated intracellularly into amastigotes. At this point, the differentiation was not complete. Hyperphosphorylation of MTOR and RPS6 suggested autophagy inhibition. d 24 h p.i. (late infection phase): amastigote differentiation was completed. ATG5, BNIP3, CTSE, MIF, UB, and miRNAs mmu-miR-155-5p and mmu-miR-210-5p, were overexpressed. Expression of miRNAs mmu-miR-101c and mmu-miR-129-5p was downregulated. The LC3B-II/LC3B-I ratio was elevated and suggested an increased autophagic flux. Glycolytic genes were upregulated. Overexpressed MIF might have attracted new uninfected host macrophages. Putative regulatory mechanisms at the RNA level were identified, which were summarized in LISA and MONA-of-LISA. Additionally, inflammatory functions (e.g., the immune response and chemokine signaling pathway) were upregulated, which indicates L. m. -infected BMDM had an inflammatory phenotype. e 48 h p.i.: amastigotes were autophagically digested, which resulted in a decline in the infection rate. p.i. = post infection
Figure Legend Snippet: Schematic summary of results. a 0 h p.i.: macrophages were infected with L. m. promastigotes. b 10 min p.i.: promastigotes attached to macrophages and were phagocytosed by the cells. c 1 h p.i. (early infection phase): promastigotes differentiated intracellularly into amastigotes. At this point, the differentiation was not complete. Hyperphosphorylation of MTOR and RPS6 suggested autophagy inhibition. d 24 h p.i. (late infection phase): amastigote differentiation was completed. ATG5, BNIP3, CTSE, MIF, UB, and miRNAs mmu-miR-155-5p and mmu-miR-210-5p, were overexpressed. Expression of miRNAs mmu-miR-101c and mmu-miR-129-5p was downregulated. The LC3B-II/LC3B-I ratio was elevated and suggested an increased autophagic flux. Glycolytic genes were upregulated. Overexpressed MIF might have attracted new uninfected host macrophages. Putative regulatory mechanisms at the RNA level were identified, which were summarized in LISA and MONA-of-LISA. Additionally, inflammatory functions (e.g., the immune response and chemokine signaling pathway) were upregulated, which indicates L. m. -infected BMDM had an inflammatory phenotype. e 48 h p.i.: amastigotes were autophagically digested, which resulted in a decline in the infection rate. p.i. = post infection

Techniques Used: Infection, Inhibition, Expressing

Global analysis of differentially expressed mRNAs in L. m. -infected BMDM and MIF western blot analyses with protein extracts from L. m. -infected and HBSS-starved BMDM. Methods: ( a – e ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium. Total RNA from 2 independent experiments was harvested from L. m. -infected BMDM or uninfected controls and hybridized with Affymetrix® chips. The BMDM were additionally incubated for 1 h in HBSS. Proteins were harvested and subjected to western blot analyses with specific antibodies against MIF. The western blots from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Results: ( a ) Differentially expressed genes were detected in L. m. -infected BMDM 1 and 24 h p.i.. The results were presented in MA plots. Large dots represent probe sets, which had significant differential expression (FDR
Figure Legend Snippet: Global analysis of differentially expressed mRNAs in L. m. -infected BMDM and MIF western blot analyses with protein extracts from L. m. -infected and HBSS-starved BMDM. Methods: ( a – e ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium. Total RNA from 2 independent experiments was harvested from L. m. -infected BMDM or uninfected controls and hybridized with Affymetrix® chips. The BMDM were additionally incubated for 1 h in HBSS. Proteins were harvested and subjected to western blot analyses with specific antibodies against MIF. The western blots from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Results: ( a ) Differentially expressed genes were detected in L. m. -infected BMDM 1 and 24 h p.i.. The results were presented in MA plots. Large dots represent probe sets, which had significant differential expression (FDR

Techniques Used: Infection, Western Blot, Mouse Assay, Incubation, Expressing

ATG5 and UB western blot analyses with protein extracts from L. m. -infected and HBSS-starved BMDM as well as determination of the infection rates of L. m. -infected BMDM after ATG5 and UB downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 and 24 h. Uninfected control BMDM were incubated for the same time in RPMI medium or starved for 1 h in HBSS. The proteins were harvested and subjected to western blot analysis with specific antibodies against ATG5 and UB. Western blots with proteins from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. c Additionally, BMDM were transfected with specific siRNAs 4 h prior to infection to downregulate the expression of ATG5 and UB. The cells were finally infected with L. m. promastigotes. L. m. -infected control BMDM were transfected with negative control siRNA. The infection rates were determined 48 h p.i.. Diagram shows the result of 2 independent experiments. Results: ( a ) ATG5 and UB levels in samples from L. m. -infected BMDM 24 h p.i. were increased compared to uninfected control BMDM. b Results of the densitometric analyses confirmed that ATG5 and UB were significantly increased in L. m. -infected BMDM 24 h p.i. compared to uninfected control BMDM. No upregulation was detected in L. m. -infected BMDM 1 h p.i. compared to the respective controls. c A significant increase in the infection rate was found in L. m. -infected BMDM after downregulation of the protein expression of ATG5 or UB compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, * p ≤ 0.05, *** p ≤ 0.001
Figure Legend Snippet: ATG5 and UB western blot analyses with protein extracts from L. m. -infected and HBSS-starved BMDM as well as determination of the infection rates of L. m. -infected BMDM after ATG5 and UB downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 and 24 h. Uninfected control BMDM were incubated for the same time in RPMI medium or starved for 1 h in HBSS. The proteins were harvested and subjected to western blot analysis with specific antibodies against ATG5 and UB. Western blots with proteins from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. c Additionally, BMDM were transfected with specific siRNAs 4 h prior to infection to downregulate the expression of ATG5 and UB. The cells were finally infected with L. m. promastigotes. L. m. -infected control BMDM were transfected with negative control siRNA. The infection rates were determined 48 h p.i.. Diagram shows the result of 2 independent experiments. Results: ( a ) ATG5 and UB levels in samples from L. m. -infected BMDM 24 h p.i. were increased compared to uninfected control BMDM. b Results of the densitometric analyses confirmed that ATG5 and UB were significantly increased in L. m. -infected BMDM 24 h p.i. compared to uninfected control BMDM. No upregulation was detected in L. m. -infected BMDM 1 h p.i. compared to the respective controls. c A significant increase in the infection rate was found in L. m. -infected BMDM after downregulation of the protein expression of ATG5 or UB compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, * p ≤ 0.05, *** p ≤ 0.001

Techniques Used: Western Blot, Infection, Mouse Assay, Incubation, Transfection, Expressing, Negative Control

27) Product Images from "Exploring regulatory networks of miR-96 in the developing inner ear"

Article Title: Exploring regulatory networks of miR-96 in the developing inner ear

Journal: Scientific Reports

doi: 10.1038/srep23363

Testing predicted targets of miR-96 and important nodes from the regulatory network. qRTPCR was carried out on cDNA from P4 organs of Corti in wildtype (green) and diminuendo homozygote (red) littermates to test gene expression changes. Error bars are standard deviation (*adjusted P
Figure Legend Snippet: Testing predicted targets of miR-96 and important nodes from the regulatory network. qRTPCR was carried out on cDNA from P4 organs of Corti in wildtype (green) and diminuendo homozygote (red) littermates to test gene expression changes. Error bars are standard deviation (*adjusted P

Techniques Used: Expressing, Standard Deviation

Testing gene expression in diminuendo homozygotes. qRTPCR was carried out on cDNA from P4 organs of Corti in wildtype (green) and diminuendo homozygote (red) littermates to test gene expression changes. Error bars are standard deviation (*adjusted P
Figure Legend Snippet: Testing gene expression in diminuendo homozygotes. qRTPCR was carried out on cDNA from P4 organs of Corti in wildtype (green) and diminuendo homozygote (red) littermates to test gene expression changes. Error bars are standard deviation (*adjusted P

Techniques Used: Expressing, Standard Deviation

28) Product Images from "Proinflammatory isoforms of IL-32 as novel and robust biomarkers for control failure in HIV-infected slow progressors"

Article Title: Proinflammatory isoforms of IL-32 as novel and robust biomarkers for control failure in HIV-infected slow progressors

Journal: Scientific Reports

doi: 10.1038/srep22902

HIV infection induces expression of IL-32 in human PBMCs. ( a ) Correlation between total IL-32 and VLs from HIV-infected SP (n = 53) (EC, VC, NVC) and TP subjects (n = 16) (subjects shown in Fig. 3c ). Spearman correlation test was used to assess the significance correlations between IL-32 and HIV VL. ( b ) Human PBMCs from n = 9 HIV-uninfected donors were either stimulated with PHA (0.25μg/ml) and IL-2 (100 units/ml) and infected with HIV-BaL (Left panel) or resting cells were infected without stimulation (Right panel). Total IL-32 was measured in the supernatant of activated cells (Left panel) or from cell lysate of non-stimulated cells (Right panel). ( c ) Total plasma IL-32 was measured in n = 10 subjects within 3 mos of HIV infection and after 1 year of ART treatment (Left panel). Total plasma IL-32 was measured in the same 10 subjects treated with ART for 1 yr and in 12 HIV neg donors (n = 12) (Right panel). The significance of between-group differences was assessed using a Wilcoxon test in panel ( b ) and the Left panel of ( c ). A Mann-Whitney test was used to assess the significance of between-group differences in panel ( c ) (Right panel).
Figure Legend Snippet: HIV infection induces expression of IL-32 in human PBMCs. ( a ) Correlation between total IL-32 and VLs from HIV-infected SP (n = 53) (EC, VC, NVC) and TP subjects (n = 16) (subjects shown in Fig. 3c ). Spearman correlation test was used to assess the significance correlations between IL-32 and HIV VL. ( b ) Human PBMCs from n = 9 HIV-uninfected donors were either stimulated with PHA (0.25μg/ml) and IL-2 (100 units/ml) and infected with HIV-BaL (Left panel) or resting cells were infected without stimulation (Right panel). Total IL-32 was measured in the supernatant of activated cells (Left panel) or from cell lysate of non-stimulated cells (Right panel). ( c ) Total plasma IL-32 was measured in n = 10 subjects within 3 mos of HIV infection and after 1 year of ART treatment (Left panel). Total plasma IL-32 was measured in the same 10 subjects treated with ART for 1 yr and in 12 HIV neg donors (n = 12) (Right panel). The significance of between-group differences was assessed using a Wilcoxon test in panel ( b ) and the Left panel of ( c ). A Mann-Whitney test was used to assess the significance of between-group differences in panel ( c ) (Right panel).

Techniques Used: Infection, Expressing, MANN-WHITNEY

29) Product Images from "Direct conversion of mouse embryonic fibroblasts into functional keratinocytes through transient expression of pluripotency-related genes"

Article Title: Direct conversion of mouse embryonic fibroblasts into functional keratinocytes through transient expression of pluripotency-related genes

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-016-0357-5

Morphological and molecular characterization of induced keratinocytes. a Induced keratinocytes ( iKCs ) exhibit keratinocyte-like morphological properties, closely resembling primary keratinocytes ( 1°KCs ) in culture. iKCs displayed a round shape and formed colonies with a cobblestone-like appearance, a characteristic of primary keratinocytes in culture. In contrast, parental mouse embryonic fibroblasts ( MEFs ) exhibit mesenchymal characteristics, as they appear spindle shaped and do not form colonies. Scale bar s = 25 μm. ( b ) Expression levels of keratinocyte and fibroblast markers were measured by semi-quantitative RT-PCR, and agarose gel electrophoresis showing the product of each PCR reaction is depicted. The relative levels of each product from three independent experiments were normalized to actin. Results are depicted as fold-change of expression compared to expression in either primary keratinocytes (K14, K5, and p63) or in MEFs (vimentin). Error bars represent standard error between experiments. In contrast to MEFs, iKCs expressed undifferentiated keratinocyte markers (K14 and K5) at levels comparable to those of primary keratinocytes; p63 expression was threefold higher in iKCs compared to MEFs, even though KCs expressed p63 at much higher levels; vimentin expression was reduced twofold in iKCs, albeit not completely abolished. c Immunofluorescence staining against vimentin ( Vim ) and keratin 14 ( K14 ) in MEFs, primary KCs, and iKCs. DAPI staining is shown in blue , and Vim or K14 staining is shown in green. Scale bars = 20 μm. ( d ) Western blot analysis to measure vimentin and K14 protein levels in MEFs and iKCs. Total cellular protein lysates were run and blotted with anti-vimentin and anti-K14 antibodies, as well as with anti-actin as a control. e Endogenous and total expression levels of pluripotency factors Klf4, Sox2, and Oct4 were assessed by RT-PCR in MEFs, KCs, and iKCs. ESCs were included as a positive control. Expression of these factors is silenced in iKCs
Figure Legend Snippet: Morphological and molecular characterization of induced keratinocytes. a Induced keratinocytes ( iKCs ) exhibit keratinocyte-like morphological properties, closely resembling primary keratinocytes ( 1°KCs ) in culture. iKCs displayed a round shape and formed colonies with a cobblestone-like appearance, a characteristic of primary keratinocytes in culture. In contrast, parental mouse embryonic fibroblasts ( MEFs ) exhibit mesenchymal characteristics, as they appear spindle shaped and do not form colonies. Scale bar s = 25 μm. ( b ) Expression levels of keratinocyte and fibroblast markers were measured by semi-quantitative RT-PCR, and agarose gel electrophoresis showing the product of each PCR reaction is depicted. The relative levels of each product from three independent experiments were normalized to actin. Results are depicted as fold-change of expression compared to expression in either primary keratinocytes (K14, K5, and p63) or in MEFs (vimentin). Error bars represent standard error between experiments. In contrast to MEFs, iKCs expressed undifferentiated keratinocyte markers (K14 and K5) at levels comparable to those of primary keratinocytes; p63 expression was threefold higher in iKCs compared to MEFs, even though KCs expressed p63 at much higher levels; vimentin expression was reduced twofold in iKCs, albeit not completely abolished. c Immunofluorescence staining against vimentin ( Vim ) and keratin 14 ( K14 ) in MEFs, primary KCs, and iKCs. DAPI staining is shown in blue , and Vim or K14 staining is shown in green. Scale bars = 20 μm. ( d ) Western blot analysis to measure vimentin and K14 protein levels in MEFs and iKCs. Total cellular protein lysates were run and blotted with anti-vimentin and anti-K14 antibodies, as well as with anti-actin as a control. e Endogenous and total expression levels of pluripotency factors Klf4, Sox2, and Oct4 were assessed by RT-PCR in MEFs, KCs, and iKCs. ESCs were included as a positive control. Expression of these factors is silenced in iKCs

Techniques Used: Expressing, Quantitative RT-PCR, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Immunofluorescence, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Positive Control

Induced keratinocytes ( iKCs ) can regenerate normal skin in vivo . ( a ) iKCs or mouse embryonic fibroblasts ( MEFs ) were transplanted in the back skin of nude mice. Nude mice transplanted with iKCs showed de novo hair formation at the graft site after 6 weeks ( a ), whereas hair development was not observed on mice transplanted with MEFs ( b ). All layers of the epidermis, including de novo hair follicles and sebaceous glands, are apparent in H E stained skin sections from iKC-transplanted mice ( c ) but not MEF-grafted skin ( d ). EYFP staining ( green ) marks iKCs and de novo iKC-generated tissue ( e ), which is not present in MEF-transplanted epidermis ( f ). Scale bars = 50 μm ( c and d ) and 10 μm ( e and f ). ( b ) Epidermal thickness comparing iKC-derived vs MEF-transplanted skin. Blindfolded comparison was performed by measuring epidermal thickness in 25 images (two measurements/image) captured from five independent tissue sections (five images/section) from iKC-derived or MEF-derived skin. Note a nearly twofold increase in iKC-generated epidermis. Error bars represent standard error amongst 50 independent measurements for each sample. ( c ) Immunofluorescence staining for K14 (a marker of undifferentiated keratinocytes), K10 (an intermediate differentiation marker), and loricrin (a terminal differentiation marker). Tissue sections were stained for different keratinocyte stratification/differentiation markers in iKC- or MEF-transplanted tissue sections. IKC-transplanted epidermis expressed both K14 ( green ) and K10 ( red ), whereas MEF-transplanted tissue only displayed some K14-positive cells, and no K10 staining ( b ). Loricrin ( red ) was expressed on the outer epidermal layer of the iKC-derived skin ( c ) and indicates the presence of iKC-derived terminally differentiated cells. In contrast, MEF-derived skin was negative for loricrin expression ( d ). Scale bars = 10 μm
Figure Legend Snippet: Induced keratinocytes ( iKCs ) can regenerate normal skin in vivo . ( a ) iKCs or mouse embryonic fibroblasts ( MEFs ) were transplanted in the back skin of nude mice. Nude mice transplanted with iKCs showed de novo hair formation at the graft site after 6 weeks ( a ), whereas hair development was not observed on mice transplanted with MEFs ( b ). All layers of the epidermis, including de novo hair follicles and sebaceous glands, are apparent in H E stained skin sections from iKC-transplanted mice ( c ) but not MEF-grafted skin ( d ). EYFP staining ( green ) marks iKCs and de novo iKC-generated tissue ( e ), which is not present in MEF-transplanted epidermis ( f ). Scale bars = 50 μm ( c and d ) and 10 μm ( e and f ). ( b ) Epidermal thickness comparing iKC-derived vs MEF-transplanted skin. Blindfolded comparison was performed by measuring epidermal thickness in 25 images (two measurements/image) captured from five independent tissue sections (five images/section) from iKC-derived or MEF-derived skin. Note a nearly twofold increase in iKC-generated epidermis. Error bars represent standard error amongst 50 independent measurements for each sample. ( c ) Immunofluorescence staining for K14 (a marker of undifferentiated keratinocytes), K10 (an intermediate differentiation marker), and loricrin (a terminal differentiation marker). Tissue sections were stained for different keratinocyte stratification/differentiation markers in iKC- or MEF-transplanted tissue sections. IKC-transplanted epidermis expressed both K14 ( green ) and K10 ( red ), whereas MEF-transplanted tissue only displayed some K14-positive cells, and no K10 staining ( b ). Loricrin ( red ) was expressed on the outer epidermal layer of the iKC-derived skin ( c ) and indicates the presence of iKC-derived terminally differentiated cells. In contrast, MEF-derived skin was negative for loricrin expression ( d ). Scale bars = 10 μm

Techniques Used: In Vivo, Mouse Assay, Staining, Generated, Derivative Assay, Immunofluorescence, Marker, Expressing

Conversion of mouse embryonic fibroblasts ( MEFs ) to functional keratinocytes ( KCs ), without isolating an induced pluripotent stem cell ( iPSC ) intermediate. a The hypothesis behind the experimental design. b After a brief initiation of pluripotency-inducing transcriptional reprogramming by transduction with Sox2, Oct4, and Klf4, induced MEFs are subsequently pushed towards the epidermal lineage by retinoic acid ( RA ) and bone morphogenetic protein-4 ( BMP-4 ) treatment. DKSF defined keratinocyte serum free, IKC induced keratinocyte
Figure Legend Snippet: Conversion of mouse embryonic fibroblasts ( MEFs ) to functional keratinocytes ( KCs ), without isolating an induced pluripotent stem cell ( iPSC ) intermediate. a The hypothesis behind the experimental design. b After a brief initiation of pluripotency-inducing transcriptional reprogramming by transduction with Sox2, Oct4, and Klf4, induced MEFs are subsequently pushed towards the epidermal lineage by retinoic acid ( RA ) and bone morphogenetic protein-4 ( BMP-4 ) treatment. DKSF defined keratinocyte serum free, IKC induced keratinocyte

Techniques Used: Functional Assay, Transduction

30) Product Images from "HIRA Is Required for Heart Development and Directly Regulates Tnni2 and Tnnt3"

Article Title: HIRA Is Required for Heart Development and Directly Regulates Tnni2 and Tnnt3

Journal: PLoS ONE

doi: 10.1371/journal.pone.0161096

HIRA is required for the regulation of a subset of genes expressed during cardiac morphogenesis. A. Venn diagram indicating the number of genes whose expression is up or downregulated in Mesp1Cre ; Hira -/fl compared to Mesp1Cre ; Hira +/fl hearts at E11.5 and E12.5. The table indicates the level of fold change observed at E11.5 and at E12.5 on a subset of genes. B. Heatmap displaying the differential gene expression in mutant (Mesp1Cre ; Hira -/fl ) vs control (Mesp1Cre ; Hira +/fl ) in triplicates at E12.5. C. Heatmap of the gene ontology analysis revealed a trend in upregulation of sarcomeric contractile fibre genes (aside from Tnnt1 and Slc4a1 which were downregulated). D. qRT-PCR and RNASeq of the indicated genes within E12.5 hearts displayed as the fold induction in the mutants compared to their WT littermates (n = 3). Unpaired t-test: p
Figure Legend Snippet: HIRA is required for the regulation of a subset of genes expressed during cardiac morphogenesis. A. Venn diagram indicating the number of genes whose expression is up or downregulated in Mesp1Cre ; Hira -/fl compared to Mesp1Cre ; Hira +/fl hearts at E11.5 and E12.5. The table indicates the level of fold change observed at E11.5 and at E12.5 on a subset of genes. B. Heatmap displaying the differential gene expression in mutant (Mesp1Cre ; Hira -/fl ) vs control (Mesp1Cre ; Hira +/fl ) in triplicates at E12.5. C. Heatmap of the gene ontology analysis revealed a trend in upregulation of sarcomeric contractile fibre genes (aside from Tnnt1 and Slc4a1 which were downregulated). D. qRT-PCR and RNASeq of the indicated genes within E12.5 hearts displayed as the fold induction in the mutants compared to their WT littermates (n = 3). Unpaired t-test: p

Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR

NKX2.5 shows diminished binding at the TTe locus in conditionally HIRA depleted hearts. NKX2.5 qChIP on 20 WT and Mesp1Cre ; Hira -/fl E12.5 hearts. Enrichment at the TTe was confirmed and found to be reduced in mutant hearts. Slc9a3r1 and Abca4 were enriched for both HIRA and NKX2.5 in WT hearts and displayed an increased expression in both Hira and Nkx2 . 5 mutant hearts. SLc9a3r1 , but not Abca4 , showed a significantly reduced binding of NKX2.5 in the absence of HIRA. Three different negative regions were tested. A region known to be enriched for NKX2.5, but not HIRA, binding ( Pop dc2) was used as a NKX2.5 qChIp positive control. Data is normalized to the input (= 100). Errors bars represent the min/max from technical duplicates. Unpaired t-test: p
Figure Legend Snippet: NKX2.5 shows diminished binding at the TTe locus in conditionally HIRA depleted hearts. NKX2.5 qChIP on 20 WT and Mesp1Cre ; Hira -/fl E12.5 hearts. Enrichment at the TTe was confirmed and found to be reduced in mutant hearts. Slc9a3r1 and Abca4 were enriched for both HIRA and NKX2.5 in WT hearts and displayed an increased expression in both Hira and Nkx2 . 5 mutant hearts. SLc9a3r1 , but not Abca4 , showed a significantly reduced binding of NKX2.5 in the absence of HIRA. Three different negative regions were tested. A region known to be enriched for NKX2.5, but not HIRA, binding ( Pop dc2) was used as a NKX2.5 qChIp positive control. Data is normalized to the input (= 100). Errors bars represent the min/max from technical duplicates. Unpaired t-test: p

Techniques Used: Binding Assay, Mutagenesis, Expressing, Positive Control

HIRA is required in the developing heart. A. Lateral view of littermate embryos with the indicated genotype. Mesp1Cre ; Hira -/fl embryos had a low penetrance external phenotype at E12.5: exencephaly (E) and light haemorrhage (H) are indicated in the mutants. At E15.5, all mutants showed severe oedema (O) as indicated. B. Transverse OPT reconstructions followed by virtual reslicing of E15.5 embryo trunks with the indicated genotype. VSD (V) and ASD (A) are indicated in the mutants. H E of transverse sections of E14.5 embryos with the indicated genotype also showed a common atrioventricular junction (CJ) in the mutants as indicated. C . H E staining of transverse sections from E12.5 embryos reveals a disruption of the endocardial cushion (EC) fusion (two controls and two littermate mutants shown). The muscular septum is deficient ( * , top) and the relatively flat rather than crescentic cushion shape in the mutant are indicated (arrows). Scale bars represent 2mm (A) , 0 . 5mm (B-C) . D . Transverse sections of E12.5 embryonic hearts of the indicated genotype immunostained with DAPI and Troponin C captured on confocal showing disrupted sarcomeric structure in the mutant ventricular free wall. Scale bar : 10μm .
Figure Legend Snippet: HIRA is required in the developing heart. A. Lateral view of littermate embryos with the indicated genotype. Mesp1Cre ; Hira -/fl embryos had a low penetrance external phenotype at E12.5: exencephaly (E) and light haemorrhage (H) are indicated in the mutants. At E15.5, all mutants showed severe oedema (O) as indicated. B. Transverse OPT reconstructions followed by virtual reslicing of E15.5 embryo trunks with the indicated genotype. VSD (V) and ASD (A) are indicated in the mutants. H E of transverse sections of E14.5 embryos with the indicated genotype also showed a common atrioventricular junction (CJ) in the mutants as indicated. C . H E staining of transverse sections from E12.5 embryos reveals a disruption of the endocardial cushion (EC) fusion (two controls and two littermate mutants shown). The muscular septum is deficient ( * , top) and the relatively flat rather than crescentic cushion shape in the mutant are indicated (arrows). Scale bars represent 2mm (A) , 0 . 5mm (B-C) . D . Transverse sections of E12.5 embryonic hearts of the indicated genotype immunostained with DAPI and Troponin C captured on confocal showing disrupted sarcomeric structure in the mutant ventricular free wall. Scale bar : 10μm .

Techniques Used: Staining, Mutagenesis

31) Product Images from "Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4"

Article Title: Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4

Journal: Scientific Reports

doi: 10.1038/srep30529

Viperin expression inhibits RABV replication. ( A ) Viperin inhibits RABV replication in viperin-eGFP-transfected BHK-21 cells. The viperin stably expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.1. Virus titres were determined at 24, 36, and 48 hpi. ( B ) RABV proteins in the infected viperin stably expressing BHK-21 cells were detected by Western blotting. ( C ) The N protein/actin, P protein/actin and M protein/actin ratios in Figure 2F were measured using Li-Cor Odyssey 3.0 analytical software version 29. ( D ) RNA expression levels of viperin. rRC-HL vRNA and N mRNA expression levels were detected by qRT-PCR at 24, 36, and 48 hpi. Viperin-expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.01. Data were normalized to β-actin expression and are presented as relative fold expression values to each control cell population infected with rRC-HL.
Figure Legend Snippet: Viperin expression inhibits RABV replication. ( A ) Viperin inhibits RABV replication in viperin-eGFP-transfected BHK-21 cells. The viperin stably expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.1. Virus titres were determined at 24, 36, and 48 hpi. ( B ) RABV proteins in the infected viperin stably expressing BHK-21 cells were detected by Western blotting. ( C ) The N protein/actin, P protein/actin and M protein/actin ratios in Figure 2F were measured using Li-Cor Odyssey 3.0 analytical software version 29. ( D ) RNA expression levels of viperin. rRC-HL vRNA and N mRNA expression levels were detected by qRT-PCR at 24, 36, and 48 hpi. Viperin-expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.01. Data were normalized to β-actin expression and are presented as relative fold expression values to each control cell population infected with rRC-HL.

Techniques Used: Expressing, Transfection, Stable Transfection, Infection, Western Blot, Software, RNA Expression, Quantitative RT-PCR

32) Product Images from "Glycyrrhizin protects against focal cerebral ischemia via inhibition of T cell activity and HMGB1-mediated mechanisms"

Article Title: Glycyrrhizin protects against focal cerebral ischemia via inhibition of T cell activity and HMGB1-mediated mechanisms

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-016-0705-5

Gly inhibits IFNγ gene expression in CD4 T cells. Leukocytes were isolated from the ischemic brain 3 days after stroke, and CD4 T cells were purified by using FACS sorter. Gene expression of IFNγ, IL-4, and IL-10 were measured by RT-qPCR. However, IL-4 and IL-10 gene expressions were not detectable. Gene expression of IFNγ was significantly inhibited by Gly administration. N = 3, * P
Figure Legend Snippet: Gly inhibits IFNγ gene expression in CD4 T cells. Leukocytes were isolated from the ischemic brain 3 days after stroke, and CD4 T cells were purified by using FACS sorter. Gene expression of IFNγ, IL-4, and IL-10 were measured by RT-qPCR. However, IL-4 and IL-10 gene expressions were not detectable. Gene expression of IFNγ was significantly inhibited by Gly administration. N = 3, * P

Techniques Used: Expressing, Isolation, Purification, FACS, Quantitative RT-PCR

33) Product Images from "FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice"

Article Title: FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0105349

Detailed analysis of adipose tissue. All data are collected from 30 weeks old mice. *indicate significant p-values between Lep ob/ob ;Fto +/+ and Lep ob/ob ; Fto −/− . a) Weights of different fat pads from female mice (n = 13, 16, 18, 16). b) Area size of epigonadal fat cells from female mice (n = 4, 4, 8, 7). c+d) Expression analysis for different marker genes of epigonadal adipose tissue (n = 6, 4, 5, 5, 5). Following p-values were calculated: between Lep ob/ob ;Fto +/− and Lep ob/ob ; Fto −/− : PPARγ2: p = 0,08, Adiponectin: p = 0,21, TNFα:p = 0,06, IL-6:p = 0,03. Data are presented as mean. Error bars indicate the SEM.
Figure Legend Snippet: Detailed analysis of adipose tissue. All data are collected from 30 weeks old mice. *indicate significant p-values between Lep ob/ob ;Fto +/+ and Lep ob/ob ; Fto −/− . a) Weights of different fat pads from female mice (n = 13, 16, 18, 16). b) Area size of epigonadal fat cells from female mice (n = 4, 4, 8, 7). c+d) Expression analysis for different marker genes of epigonadal adipose tissue (n = 6, 4, 5, 5, 5). Following p-values were calculated: between Lep ob/ob ;Fto +/− and Lep ob/ob ; Fto −/− : PPARγ2: p = 0,08, Adiponectin: p = 0,21, TNFα:p = 0,06, IL-6:p = 0,03. Data are presented as mean. Error bars indicate the SEM.

Techniques Used: Mouse Assay, Expressing, Marker

34) Product Images from "Oncogene-triggered suppression of DNA repair leads to DNA instability in cancer"

Article Title: Oncogene-triggered suppression of DNA repair leads to DNA instability in cancer

Journal: Oncotarget

doi:

NeuT oncogene suppresses expression of H2AX A Expression of NeuT in MCF10A cells. MCF10A cells were infected with NeuT- expressing virus and two days post-infection selected with puromicin (0.75μg/ml). Control cells were infected with a corresponding empty virus. After 6 days of selection, cells were harvested, lysed, and analyzed by immunoblotting. B. NeuT expression does not affect stability of H2AX. MCF10A cells were infected as above. Cells were treated with emetine (10μM) and levels of H2AX at corresponding time points were assayed by immunoblotting. C. NeuT expression doesn't suppress translation of H2AX mRNA. MCF10A cells were infected as above. On day 6, post-infection cells were transfected with plasmid expressing renilla luciferase under H2AX 3'UTR control and the firefly luciferase used as an internal control. Translational efficiency was measured as renilla/luciferase ration (relative units). D. NeuT oncogene suppresses transcription of H2AX mRNA. MCF10A cells were infected as above, and Q-PCR was performed as described in Materials and Methods to measure H2AX mRNA. Each sample was analyzed in triplicates. E. NeuT expression does not affect stability of H2AX mRNA. MCF10A cells were infected as above. Cells were treated with actinomycin D (7μg/ml) and samples for RNA extraction were collected at corresponding time points. Q-PCR was performed as described in Materials and Methods. Each point represents three independent experiments. F. Inhibition of CDKs suppresses expression of H2AX. MCF10A cells were treated with indicated doses of roscovitine overnight. Cells were lysed and H2AX levels were assayed by immunoblotting. G. Rb silencing reverses suppression of H2AX levels caused by expression of NeuT. MCF10A cells were simultaneously infected with NeuT- expressing retrovirus (under blasticidin selection) and siRB1-expressing lentivirus (under puromycin selection); control cells were infected with corresponding empty viruses. Two days post-infection puromycin (0.75μg/ml) and blastocidin (10μg/ml) were added and after another 7 days cells were harvested, lysed, and analyzed by immunoblotting to assay H2AX levels (left panel) or by Q-PCR to assay level of Rb1 silencing (right panel).
Figure Legend Snippet: NeuT oncogene suppresses expression of H2AX A Expression of NeuT in MCF10A cells. MCF10A cells were infected with NeuT- expressing virus and two days post-infection selected with puromicin (0.75μg/ml). Control cells were infected with a corresponding empty virus. After 6 days of selection, cells were harvested, lysed, and analyzed by immunoblotting. B. NeuT expression does not affect stability of H2AX. MCF10A cells were infected as above. Cells were treated with emetine (10μM) and levels of H2AX at corresponding time points were assayed by immunoblotting. C. NeuT expression doesn't suppress translation of H2AX mRNA. MCF10A cells were infected as above. On day 6, post-infection cells were transfected with plasmid expressing renilla luciferase under H2AX 3'UTR control and the firefly luciferase used as an internal control. Translational efficiency was measured as renilla/luciferase ration (relative units). D. NeuT oncogene suppresses transcription of H2AX mRNA. MCF10A cells were infected as above, and Q-PCR was performed as described in Materials and Methods to measure H2AX mRNA. Each sample was analyzed in triplicates. E. NeuT expression does not affect stability of H2AX mRNA. MCF10A cells were infected as above. Cells were treated with actinomycin D (7μg/ml) and samples for RNA extraction were collected at corresponding time points. Q-PCR was performed as described in Materials and Methods. Each point represents three independent experiments. F. Inhibition of CDKs suppresses expression of H2AX. MCF10A cells were treated with indicated doses of roscovitine overnight. Cells were lysed and H2AX levels were assayed by immunoblotting. G. Rb silencing reverses suppression of H2AX levels caused by expression of NeuT. MCF10A cells were simultaneously infected with NeuT- expressing retrovirus (under blasticidin selection) and siRB1-expressing lentivirus (under puromycin selection); control cells were infected with corresponding empty viruses. Two days post-infection puromycin (0.75μg/ml) and blastocidin (10μg/ml) were added and after another 7 days cells were harvested, lysed, and analyzed by immunoblotting to assay H2AX levels (left panel) or by Q-PCR to assay level of Rb1 silencing (right panel).

Techniques Used: Expressing, Infection, Selection, Transfection, Plasmid Preparation, Luciferase, Polymerase Chain Reaction, RNA Extraction, Inhibition

35) Product Images from "Effects of Cdh23 single nucleotide substitutions on age-related hearing loss in C57BL/6 and 129S1/Sv mice and comparisons with congenic strains"

Article Title: Effects of Cdh23 single nucleotide substitutions on age-related hearing loss in C57BL/6 and 129S1/Sv mice and comparisons with congenic strains

Journal: Scientific Reports

doi: 10.1038/srep44450

DNA sequence validation, PCR identification of targeted SNVs and assessment of exon skpping. ( A ) Sequence chromatograms of PCR amplified DNA surrounding the targeted Cdh23 c.753 nucleotide (indicated by the red downward-pointing arrow) confirm that C57BL/6 NJ (B6N) and 129S- Cdh23 c.753A (129S-SNV) mice are homozygous for the Cdh23 c.753 A nucleotide, while 129S1/SvImJ (129S1) and B6N- Cdh23 c.753G (B6N-SNV) mice are homozygous for the Cdh23 c.753 G nucleotide. ( B ) Identification of Cdh23 alleles with targeted SNVs by PCR amplification of the closely linked PGK-Neo insertion remnant. Primers flanking the PGK-Neo cassette insertion site were used to amplify PCR products that differ in size between the wildtype allele and the targeted SNV allele, which retains an intronic 104 bp remnant of the PGK-Neo cassette after Cre deletion. Because of its close proximity (178 bp) to the targeted SNV, the presence or absence of the PGK-Neo remnant can be used to distinguish the wildtype allele (+, 112 bp) from the targeted SNV allele ( v , 216 bp). Lane 1, 100-bp DNA size ladder; lanes 2–5, genotypes of individual mice. ( C ) RT-PCR to evaluate the extent of exon skipping related to the Cdh23 c.753A variant. cDNA primers flanking the alternatively spliced exon of Cdh23 (containing the c.753 nucleotide) were used to amplify alternatively spliced products. The PCR product size from the wild-type Cdh23 transcript (395 bp) is larger than the PCR product from the alternatively spliced, in-frame transcript (266 bp), which lacks the 129 bp skipped exon. Lane 1, 100-bp DNA size ladder; lane 2, B6N; lane 3, 129S1; lane 4, B6.129S1- Cdh23 Ahl + congenic (B6-con); lane 5, 129S1.B6- Cdh23 ahl congenic (129S-con); lane 6, B6- Cdh23 c.753G SNV (B6-SNV); and lane 7, 129S- Cdh23 c.753A SNV (129S-SNV). Lanes 2, 5, and 7 show alternatively spliced transcripts caused by the Cdh23 c.753A variant.
Figure Legend Snippet: DNA sequence validation, PCR identification of targeted SNVs and assessment of exon skpping. ( A ) Sequence chromatograms of PCR amplified DNA surrounding the targeted Cdh23 c.753 nucleotide (indicated by the red downward-pointing arrow) confirm that C57BL/6 NJ (B6N) and 129S- Cdh23 c.753A (129S-SNV) mice are homozygous for the Cdh23 c.753 A nucleotide, while 129S1/SvImJ (129S1) and B6N- Cdh23 c.753G (B6N-SNV) mice are homozygous for the Cdh23 c.753 G nucleotide. ( B ) Identification of Cdh23 alleles with targeted SNVs by PCR amplification of the closely linked PGK-Neo insertion remnant. Primers flanking the PGK-Neo cassette insertion site were used to amplify PCR products that differ in size between the wildtype allele and the targeted SNV allele, which retains an intronic 104 bp remnant of the PGK-Neo cassette after Cre deletion. Because of its close proximity (178 bp) to the targeted SNV, the presence or absence of the PGK-Neo remnant can be used to distinguish the wildtype allele (+, 112 bp) from the targeted SNV allele ( v , 216 bp). Lane 1, 100-bp DNA size ladder; lanes 2–5, genotypes of individual mice. ( C ) RT-PCR to evaluate the extent of exon skipping related to the Cdh23 c.753A variant. cDNA primers flanking the alternatively spliced exon of Cdh23 (containing the c.753 nucleotide) were used to amplify alternatively spliced products. The PCR product size from the wild-type Cdh23 transcript (395 bp) is larger than the PCR product from the alternatively spliced, in-frame transcript (266 bp), which lacks the 129 bp skipped exon. Lane 1, 100-bp DNA size ladder; lane 2, B6N; lane 3, 129S1; lane 4, B6.129S1- Cdh23 Ahl + congenic (B6-con); lane 5, 129S1.B6- Cdh23 ahl congenic (129S-con); lane 6, B6- Cdh23 c.753G SNV (B6-SNV); and lane 7, 129S- Cdh23 c.753A SNV (129S-SNV). Lanes 2, 5, and 7 show alternatively spliced transcripts caused by the Cdh23 c.753A variant.

Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Variant Assay

36) Product Images from "Defective protein repair under methionine sulfoxide A deletion drives autophagy and ARE-dependent gene transcription"

Article Title: Defective protein repair under methionine sulfoxide A deletion drives autophagy and ARE-dependent gene transcription

Journal: Redox Biology

doi: 10.1016/j.redox.2018.04.001

Expression and activity of Nrf2-regulated genes are elevated under MsrA deletion. (A, B) GCLC (A) and GR (B) mRNA levels in MsrA-/- and WT VSMC by qRT-PCR, normalized to ARP; n = 3 biological replicates. (C-E) Representative immunoblot (C) and summary data for GCLC (D) and GR (E) protein levels in whole cell lysates from MsrA-/- and WT VSMC. Data were normalized to GAPDH; n = 6–7 biological replicates. (F) GR activity, normalized to total protein concentration; n = 4 biological replicates. (G, H) Levels of reduced glutathione (GSH, G) and oxidized glutathione (GSSG, H), normalized to total protein; n = 4 biological replicates. (I) Ratio of reduced/oxidized glutathione. (J) Glutathione transferase (GST) activity, normalized to total protein; n = 7 biological replicates. * p
Figure Legend Snippet: Expression and activity of Nrf2-regulated genes are elevated under MsrA deletion. (A, B) GCLC (A) and GR (B) mRNA levels in MsrA-/- and WT VSMC by qRT-PCR, normalized to ARP; n = 3 biological replicates. (C-E) Representative immunoblot (C) and summary data for GCLC (D) and GR (E) protein levels in whole cell lysates from MsrA-/- and WT VSMC. Data were normalized to GAPDH; n = 6–7 biological replicates. (F) GR activity, normalized to total protein concentration; n = 4 biological replicates. (G, H) Levels of reduced glutathione (GSH, G) and oxidized glutathione (GSSG, H), normalized to total protein; n = 4 biological replicates. (I) Ratio of reduced/oxidized glutathione. (J) Glutathione transferase (GST) activity, normalized to total protein; n = 7 biological replicates. * p

Techniques Used: Expressing, Activity Assay, Quantitative RT-PCR, Protein Concentration

p62 is elevated in MsrA-deficient VSMC and arteries. (A, B) Representative immunoblot (A) and summary data (B) for p62 in whole cell lysates of VSMC isolated from MsrA-/- and WT mice. Data were normalized to GAPDH loading control and expressed relative to p62 levels in WT VSMC (n = 9 biological replicates). (C, D) Representative immunoblot (C) and summary data (D) for p62 in whole cell lysates of carotid arteries isolated from MsrA-/- and WT mice (n = 10 biological replicates). E) Representative immunofluorescent images of p62 (green) and nuclei (TOPRO, blue) in VSMC from MsrA-/- and WT mice with or without treatment with bafilomycin a1 (Baf) for 24 h. Scale bars 20 µm. (F) Quantification of p62 aggregates from (E). Arbitrary aggregate score was calculated as the mean GFP fluorescence intensity per cell in at least 5 images per biological replicate (1–5 cells/image; n = 5 biological replicates). (G) Representative immunofluorescent images of p62 (green), smooth muscle actin (red) and nuclei (TOPRO, blue) in carotid artery sections from MsrA-/- and WT mice. 100 × , scale bar 10 µm. NC denotes negative control without primary antibody, p62 inset with p62 (green) only. Arrows denote p62 aggregates. (H) mRNA expression of p62 in VSMC from MsrA-/- and WT mice by qRT-PCR; data were normalized to ARP and expressed relative to p62 in WT VSMC (n = 5 biological replicates). * p
Figure Legend Snippet: p62 is elevated in MsrA-deficient VSMC and arteries. (A, B) Representative immunoblot (A) and summary data (B) for p62 in whole cell lysates of VSMC isolated from MsrA-/- and WT mice. Data were normalized to GAPDH loading control and expressed relative to p62 levels in WT VSMC (n = 9 biological replicates). (C, D) Representative immunoblot (C) and summary data (D) for p62 in whole cell lysates of carotid arteries isolated from MsrA-/- and WT mice (n = 10 biological replicates). E) Representative immunofluorescent images of p62 (green) and nuclei (TOPRO, blue) in VSMC from MsrA-/- and WT mice with or without treatment with bafilomycin a1 (Baf) for 24 h. Scale bars 20 µm. (F) Quantification of p62 aggregates from (E). Arbitrary aggregate score was calculated as the mean GFP fluorescence intensity per cell in at least 5 images per biological replicate (1–5 cells/image; n = 5 biological replicates). (G) Representative immunofluorescent images of p62 (green), smooth muscle actin (red) and nuclei (TOPRO, blue) in carotid artery sections from MsrA-/- and WT mice. 100 × , scale bar 10 µm. NC denotes negative control without primary antibody, p62 inset with p62 (green) only. Arrows denote p62 aggregates. (H) mRNA expression of p62 in VSMC from MsrA-/- and WT mice by qRT-PCR; data were normalized to ARP and expressed relative to p62 in WT VSMC (n = 5 biological replicates). * p

Techniques Used: Isolation, Mouse Assay, Fluorescence, Negative Control, Expressing, Quantitative RT-PCR

Elevated Nrf2 protein expression under MsrA deficiency is due to protein stabilization rather than increased transcription. (A, B) Representative immunoblot (A) and quantification (B) for Nrf2 protein levels in whole cell lysates from MsrA-/- and WT VSMC; n = 3 biological replicates. (C) Nrf2 mRNA levels in VSMC by qRT-PCR; n = 5 biological replicates. (D) Representative immunoprecipitation of Nrf2 followed by immunoblot for ubiquitin in MsrA-/- and WT VSMC. IgG: IP with IgG, WT + MC132: IP with anti-Nrf2 in WT VSMC incubated with MG132, WCL: whole cell lysate of WT VSMC as controls. (E) Quantification of (D); n = 7 biological replicates. (F) p62 mRNA levels by qRT-PCR in aortic samples WT, MsrA-/- and MsrA-/- x Nrf2-/- mice; n = 7, 9 biological replicates. (G) Representative Immunoblots for Nrf2 and GAPDH in aortic samples from WT, MsrA-/- and MsrA-/- x Nrf2-/- mice. (H) Quantification of (G) n = 7 biological replicates. (E) * p
Figure Legend Snippet: Elevated Nrf2 protein expression under MsrA deficiency is due to protein stabilization rather than increased transcription. (A, B) Representative immunoblot (A) and quantification (B) for Nrf2 protein levels in whole cell lysates from MsrA-/- and WT VSMC; n = 3 biological replicates. (C) Nrf2 mRNA levels in VSMC by qRT-PCR; n = 5 biological replicates. (D) Representative immunoprecipitation of Nrf2 followed by immunoblot for ubiquitin in MsrA-/- and WT VSMC. IgG: IP with IgG, WT + MC132: IP with anti-Nrf2 in WT VSMC incubated with MG132, WCL: whole cell lysate of WT VSMC as controls. (E) Quantification of (D); n = 7 biological replicates. (F) p62 mRNA levels by qRT-PCR in aortic samples WT, MsrA-/- and MsrA-/- x Nrf2-/- mice; n = 7, 9 biological replicates. (G) Representative Immunoblots for Nrf2 and GAPDH in aortic samples from WT, MsrA-/- and MsrA-/- x Nrf2-/- mice. (H) Quantification of (G) n = 7 biological replicates. (E) * p

Techniques Used: Expressing, Quantitative RT-PCR, Immunoprecipitation, Incubation, Mouse Assay, Western Blot

37) Product Images from "Histone chaperone HIRA deposits histone H3.3 onto foreign viral DNA and contributes to anti-viral intrinsic immunity"

Article Title: Histone chaperone HIRA deposits histone H3.3 onto foreign viral DNA and contributes to anti-viral intrinsic immunity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx771

HIRA contributes to efficient suppression of viral infection. ( A ) Virus yield from HIRA-depleted and control IMR90 cells infected at MOI 0.01 with ICP0-null HSV-1 mutant dl 1403 CMV lacZ or wt HSV-1 variant in 1863. Supernatant was harvested at indicated times post infection (h p.i.) and virus titres determined by plaque assay. Data are mean +/- SD (error bars) (n = 3 biological repeats) with indicated P values. ( B ) HIRA-depleted and control IMR90 cells infected with ICP0-null HSV-1 mutant dl 1403 at MOI 2.0. Lysates were harvested and processed at indicated time points post infection (h p.i.) ( C – E ) Control ( CAGG-Cre-ER , WT, +tamoxifen) or Hira-deficient ( CAGG-Cre-ER, Hirafl/fl , +tamoxifen) mice were infected with MCMV and spleen harvested 4 days later. Each spleen was divided into three pieces for downstream analysis. (C) Western blot analysis of WT or HIRA −/− animals showing knock out of Hira. Shown are representative western blot results from two different gels with 4 WT and 8 HIRA −/- mice. (D) mRNA abundance of IFN-β target genes by qRT-PCR. Bar chart displays mean of each IFN-β target gene mRNA abundance in WT mice compared to HIRA −/- mice, normalized to β - actin as housekeeping control. Data are mean ± SEM (error bars) ( n = 4 for WT mice and n = 8 for HIRA −/- mice). * P
Figure Legend Snippet: HIRA contributes to efficient suppression of viral infection. ( A ) Virus yield from HIRA-depleted and control IMR90 cells infected at MOI 0.01 with ICP0-null HSV-1 mutant dl 1403 CMV lacZ or wt HSV-1 variant in 1863. Supernatant was harvested at indicated times post infection (h p.i.) and virus titres determined by plaque assay. Data are mean +/- SD (error bars) (n = 3 biological repeats) with indicated P values. ( B ) HIRA-depleted and control IMR90 cells infected with ICP0-null HSV-1 mutant dl 1403 at MOI 2.0. Lysates were harvested and processed at indicated time points post infection (h p.i.) ( C – E ) Control ( CAGG-Cre-ER , WT, +tamoxifen) or Hira-deficient ( CAGG-Cre-ER, Hirafl/fl , +tamoxifen) mice were infected with MCMV and spleen harvested 4 days later. Each spleen was divided into three pieces for downstream analysis. (C) Western blot analysis of WT or HIRA −/− animals showing knock out of Hira. Shown are representative western blot results from two different gels with 4 WT and 8 HIRA −/- mice. (D) mRNA abundance of IFN-β target genes by qRT-PCR. Bar chart displays mean of each IFN-β target gene mRNA abundance in WT mice compared to HIRA −/- mice, normalized to β - actin as housekeeping control. Data are mean ± SEM (error bars) ( n = 4 for WT mice and n = 8 for HIRA −/- mice). * P

Techniques Used: Infection, Mutagenesis, Variant Assay, Plaque Assay, Mouse Assay, Western Blot, Knock-Out, Quantitative RT-PCR

38) Product Images from "Prolactin signaling through focal adhesion complexes is amplified by stiff extracellular matrices in breast cancer cells"

Article Title: Prolactin signaling through focal adhesion complexes is amplified by stiff extracellular matrices in breast cancer cells

Journal: Oncotarget

doi: 10.18632/oncotarget.10137

Inhibiting SFKs decreases PRL signals to pFAK Y925 only in stiff environments A-B. T47D cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24h, then treated with vehicle (−) or SFK inhibitor, PP-2 (+) for 1 h prior to ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 3. Different letters represent significant differences within each stiffness determined by paired t-tests (lower case, 12kPa; upper case, 75kPa), p
Figure Legend Snippet: Inhibiting SFKs decreases PRL signals to pFAK Y925 only in stiff environments A-B. T47D cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24h, then treated with vehicle (−) or SFK inhibitor, PP-2 (+) for 1 h prior to ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 3. Different letters represent significant differences within each stiffness determined by paired t-tests (lower case, 12kPa; upper case, 75kPa), p

Techniques Used: Western Blot

Collagen ligand density does not modulate PRL signals to ERK1/2 or FAK A-C. T47D were cells plated on 25 kPa polyacrylamide gels coated with either 50, 200, or 800 μg/ml collagen-I, serum starved for 24h, then treated ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 3. Different letters represent significant differences between treatments, p
Figure Legend Snippet: Collagen ligand density does not modulate PRL signals to ERK1/2 or FAK A-C. T47D were cells plated on 25 kPa polyacrylamide gels coated with either 50, 200, or 800 μg/ml collagen-I, serum starved for 24h, then treated ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 3. Different letters represent significant differences between treatments, p

Techniques Used: Western Blot

Blocking β1-integrin decreases PRL signals to pERK1/2 and pFAK Y925 in stiff environments A-B. T47D cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24h, then treated with isotype control antibody (−) or β1-integrin blocking antibody mAb13 (+) for 1 h prior to ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M., n = 4. Different letters represent significant differences within each stiffness (lower case, 12kPa; upper case, 75kPa), p
Figure Legend Snippet: Blocking β1-integrin decreases PRL signals to pERK1/2 and pFAK Y925 in stiff environments A-B. T47D cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24h, then treated with isotype control antibody (−) or β1-integrin blocking antibody mAb13 (+) for 1 h prior to ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M., n = 4. Different letters represent significant differences within each stiffness (lower case, 12kPa; upper case, 75kPa), p

Techniques Used: Blocking Assay, Western Blot

Inhibiting integrin activated FAK at Y397 more efficiently decreases PRL signals to pFAK Y925 in stiff environments A-B. T47D cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24h, then treated with vehicle (−) or FAK Y397 inhibitor PF-573228 (+) for 1 h prior to ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 4. Different letters represent significant differences within each stiffness (lower case, 12kPa; upper case, 75kPa), p
Figure Legend Snippet: Inhibiting integrin activated FAK at Y397 more efficiently decreases PRL signals to pFAK Y925 in stiff environments A-B. T47D cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24h, then treated with vehicle (−) or FAK Y397 inhibitor PF-573228 (+) for 1 h prior to ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 4. Different letters represent significant differences within each stiffness (lower case, 12kPa; upper case, 75kPa), p

Techniques Used: Western Blot

Stiff environments increase FAK-mediated hormone induced proliferation T47D and MCF-7 cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I in phenol-red free 5% charcoal stripped FBS for 24 h, serum starved for 24 h, and then treated with vehicle (DMSO 1:1000) or the FAK inhibitor, PF-573228 (1μM), for 1 h prior to ± PRL (4 nM), ± E2 (1nM) for 24 h. Cells were then stained with DAPI and Ki-67 antibody as described in Experimental Procedures. A, C. Effect of hormones on Ki67 staining, assessed by percentage of Ki-67 positive T47D (A) and MCF-7 (C) cells. B, D. Inhibition of proliferation by PF-573,228 compared to vehicle treated T47D cells (B) and MCF7 cells (D). Different letters represent significant differences within each stiffness (lower case, 12 kPa; upper case, 75 kPa). * represent significant differences between the same treatments at different stiffnesses: *p
Figure Legend Snippet: Stiff environments increase FAK-mediated hormone induced proliferation T47D and MCF-7 cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I in phenol-red free 5% charcoal stripped FBS for 24 h, serum starved for 24 h, and then treated with vehicle (DMSO 1:1000) or the FAK inhibitor, PF-573228 (1μM), for 1 h prior to ± PRL (4 nM), ± E2 (1nM) for 24 h. Cells were then stained with DAPI and Ki-67 antibody as described in Experimental Procedures. A, C. Effect of hormones on Ki67 staining, assessed by percentage of Ki-67 positive T47D (A) and MCF-7 (C) cells. B, D. Inhibition of proliferation by PF-573,228 compared to vehicle treated T47D cells (B) and MCF7 cells (D). Different letters represent significant differences within each stiffness (lower case, 12 kPa; upper case, 75 kPa). * represent significant differences between the same treatments at different stiffnesses: *p

Techniques Used: Staining, Inhibition

Stiffer environments robustly increase PRL signals to pERK1/2 and pFAK Y925, but only slightly increase signals to pSTAT5 A-C. T47D cells were plated on 12, 25, or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24 h, and treated ± PRL (4nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 5. Different letters represent significant differences between treatments, p
Figure Legend Snippet: Stiffer environments robustly increase PRL signals to pERK1/2 and pFAK Y925, but only slightly increase signals to pSTAT5 A-C. T47D cells were plated on 12, 25, or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24 h, and treated ± PRL (4nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 5. Different letters represent significant differences between treatments, p

Techniques Used: Western Blot

39) Product Images from "Changes of circulating Th22 cells in children with hand, foot, and mouth disease caused by enterovirus 71 infection"

Article Title: Changes of circulating Th22 cells in children with hand, foot, and mouth disease caused by enterovirus 71 infection

Journal: Oncotarget

doi: 10.18632/oncotarget.14083

Increased mRNA expression of cTh22 cells is associated with cytokines and transcription factors in patients with EV71-associated HFMD A .- C . Human peripheral blood mononuclear cells (PBMCs) from 18 mild patients and 11 severe patients with EV71-associated HFMD and 11 healthy controls (HC) were isolated and analyzed using the PCR assay for IL-6, IL-23 and TNF-α mRNA expression. D .- G . The mRNA levels of IL-22, IL-17A, AHR and RORγt in CD4 + T cells were analyzed using the PCR assay as described in the Methods section. *, p
Figure Legend Snippet: Increased mRNA expression of cTh22 cells is associated with cytokines and transcription factors in patients with EV71-associated HFMD A .- C . Human peripheral blood mononuclear cells (PBMCs) from 18 mild patients and 11 severe patients with EV71-associated HFMD and 11 healthy controls (HC) were isolated and analyzed using the PCR assay for IL-6, IL-23 and TNF-α mRNA expression. D .- G . The mRNA levels of IL-22, IL-17A, AHR and RORγt in CD4 + T cells were analyzed using the PCR assay as described in the Methods section. *, p

Techniques Used: Expressing, Isolation, Polymerase Chain Reaction

40) Product Images from "Changes of circulating Th22 cells in children with hand, foot, and mouth disease caused by enterovirus 71 infection"

Article Title: Changes of circulating Th22 cells in children with hand, foot, and mouth disease caused by enterovirus 71 infection

Journal: Oncotarget

doi: 10.18632/oncotarget.14083

Circulating Th22 cells, a principal source of intracellular IL-22 in peripheral blood A . The cells were gated initially on IL-22 + cells and then on CD4 + T cells and IL-17A cells. B . Circulating IL-22 + IL-17A − CD4 + Th22 (cTh22) cells. C . Circulating IL-22 + cTh17 cells. D . IL-17A − CD4 − cells in the total IL-22 + cells were analyzed in HC and mild and severe patients. *, p
Figure Legend Snippet: Circulating Th22 cells, a principal source of intracellular IL-22 in peripheral blood A . The cells were gated initially on IL-22 + cells and then on CD4 + T cells and IL-17A cells. B . Circulating IL-22 + IL-17A − CD4 + Th22 (cTh22) cells. C . Circulating IL-22 + cTh17 cells. D . IL-17A − CD4 − cells in the total IL-22 + cells were analyzed in HC and mild and severe patients. *, p

Techniques Used:

Increased frequencies of cTh22 and cTh17 cells in human CD4 + T cells of peripheral blood from patients with EV71-associated HFMD Human peripheral blood mononuclear cells (PBMCs) from 32 mild and 24 severe patients with EV71-associated HFMD and 46 healthy controls (HC) were isolated and stained with labeled antibodies and analyzed by flow cytometry as described in the Methods section. A . The cells were gated initially on lymphocytes and then on CD4 + T cells, circulating IL-22 + IL-17A − CD4 + Th22 (cTh22) cells, circulating IL-17A + CD4 + Th17 (cTh17) cells, and IL-22 + IL-17A + CD4 + T (IL-22 + cTh17)cells in the total number of CD4 + T cells. B .- D . The analysis of the percentages of cTh22 cells, cTh17 cells and IL-22 + cTh17 cells in CD4 + T cells from HC and mild and severe patients. E .- F . The proportion of circulating IL-22 + cells in cTh17 cells. G .- H . The correlation of cTh17 cells and IL-22 + cTh17 cells in mild and severe cases. *, p
Figure Legend Snippet: Increased frequencies of cTh22 and cTh17 cells in human CD4 + T cells of peripheral blood from patients with EV71-associated HFMD Human peripheral blood mononuclear cells (PBMCs) from 32 mild and 24 severe patients with EV71-associated HFMD and 46 healthy controls (HC) were isolated and stained with labeled antibodies and analyzed by flow cytometry as described in the Methods section. A . The cells were gated initially on lymphocytes and then on CD4 + T cells, circulating IL-22 + IL-17A − CD4 + Th22 (cTh22) cells, circulating IL-17A + CD4 + Th17 (cTh17) cells, and IL-22 + IL-17A + CD4 + T (IL-22 + cTh17)cells in the total number of CD4 + T cells. B .- D . The analysis of the percentages of cTh22 cells, cTh17 cells and IL-22 + cTh17 cells in CD4 + T cells from HC and mild and severe patients. E .- F . The proportion of circulating IL-22 + cells in cTh17 cells. G .- H . The correlation of cTh17 cells and IL-22 + cTh17 cells in mild and severe cases. *, p

Techniques Used: Isolation, Staining, Labeling, Flow Cytometry, Cytometry

Increased mRNA expression of cTh22 cells is associated with cytokines and transcription factors in patients with EV71-associated HFMD A .- C . Human peripheral blood mononuclear cells (PBMCs) from 18 mild patients and 11 severe patients with EV71-associated HFMD and 11 healthy controls (HC) were isolated and analyzed using the PCR assay for IL-6, IL-23 and TNF-α mRNA expression. D .- G . The mRNA levels of IL-22, IL-17A, AHR and RORγt in CD4 + T cells were analyzed using the PCR assay as described in the Methods section. *, p
Figure Legend Snippet: Increased mRNA expression of cTh22 cells is associated with cytokines and transcription factors in patients with EV71-associated HFMD A .- C . Human peripheral blood mononuclear cells (PBMCs) from 18 mild patients and 11 severe patients with EV71-associated HFMD and 11 healthy controls (HC) were isolated and analyzed using the PCR assay for IL-6, IL-23 and TNF-α mRNA expression. D .- G . The mRNA levels of IL-22, IL-17A, AHR and RORγt in CD4 + T cells were analyzed using the PCR assay as described in the Methods section. *, p

Techniques Used: Expressing, Isolation, Polymerase Chain Reaction

Related Articles

Clone Assay:

Article Title: BAK/BAX-Mediated Apoptosis Is a Myc-Induced Roadblock to Reprogramming
Article Snippet: .. Genomic DNA was isolated from six clones of each genotype, plus the corresponding MEFs from which the clones were derived, using a DNeasy blood and tissue kit (Qiagen). .. Libraries were prepared from sheared genomic DNA (∼300 bp; 4 ng per sample) using a Truseq DNA sample preparation kit (Illumina).

RNA Extraction:

Article Title: Transposon mutagenesis screen in mice identifies TM9SF2 as a novel colorectal cancer oncogene
Article Snippet: .. RNA extraction and qRT-PCR Total RNA was extracted using the RNeasy Plus mini kit (Qiagen, Germantown, MD) according to the manufacturer’s protocol. .. Total RNA concentration and quality were measured using the BioTek Epoch microplate spectrophotometer (BioTek, Winooski, VT).

RNA Sequencing Assay:

Article Title: The loss of SMG1 causes defects in quality control pathways in Physcomitrella patens
Article Snippet: .. RNA Isolation, RNA-seq and qRT-PCR Total RNA was collected from 100 mg of protonema tissue, five-days post homogenisation using the RNeasy Plant Mini Kit with on-column DNase I treatment (Qiagen). .. RNA for RNA-seq was sent to GTAC Biotech (Constance, Germany) for library preparation using the TruSeq sample preparation kit (Illumina) and sequencing on the Illumina HiSeq2000 to generate single-end 100 nt long reads.

Homogenization:

Article Title: The loss of SMG1 causes defects in quality control pathways in Physcomitrella patens
Article Snippet: .. RNA Isolation, RNA-seq and qRT-PCR Total RNA was collected from 100 mg of protonema tissue, five-days post homogenisation using the RNeasy Plant Mini Kit with on-column DNase I treatment (Qiagen). .. RNA for RNA-seq was sent to GTAC Biotech (Constance, Germany) for library preparation using the TruSeq sample preparation kit (Illumina) and sequencing on the Illumina HiSeq2000 to generate single-end 100 nt long reads.

Isolation:

Article Title: BAK/BAX-Mediated Apoptosis Is a Myc-Induced Roadblock to Reprogramming
Article Snippet: .. Genomic DNA was isolated from six clones of each genotype, plus the corresponding MEFs from which the clones were derived, using a DNeasy blood and tissue kit (Qiagen). .. Libraries were prepared from sheared genomic DNA (∼300 bp; 4 ng per sample) using a Truseq DNA sample preparation kit (Illumina).

Article Title: Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632
Article Snippet: .. After removal of the feeder cells with Versene, RNA from SBK at passage 5 and 6 for the +Y group and 4, 5, and 6 for the -Y group was isolated using the RNeasy kit (Qiagen). ..

Article Title: The loss of SMG1 causes defects in quality control pathways in Physcomitrella patens
Article Snippet: .. RNA Isolation, RNA-seq and qRT-PCR Total RNA was collected from 100 mg of protonema tissue, five-days post homogenisation using the RNeasy Plant Mini Kit with on-column DNase I treatment (Qiagen). .. RNA for RNA-seq was sent to GTAC Biotech (Constance, Germany) for library preparation using the TruSeq sample preparation kit (Illumina) and sequencing on the Illumina HiSeq2000 to generate single-end 100 nt long reads.

Quantitative RT-PCR:

Article Title: Transposon mutagenesis screen in mice identifies TM9SF2 as a novel colorectal cancer oncogene
Article Snippet: .. RNA extraction and qRT-PCR Total RNA was extracted using the RNeasy Plus mini kit (Qiagen, Germantown, MD) according to the manufacturer’s protocol. .. Total RNA concentration and quality were measured using the BioTek Epoch microplate spectrophotometer (BioTek, Winooski, VT).

Article Title: mTORC1 is a key mediator of RON-dependent breast cancer metastasis with therapeutic potential
Article Snippet: .. qRT-PCR Total RNA from T47D-RON WT and mutants was extracted with the Qiagen RNeasy Mini kit (Qiagen), according to the manufacturer’s instructions. .. 500 ng of purified RNA from each sample was reverse transcribed using SuperScript first-strand synthesis system for RT-PCR (Invitrogen).

Article Title: The loss of SMG1 causes defects in quality control pathways in Physcomitrella patens
Article Snippet: .. RNA Isolation, RNA-seq and qRT-PCR Total RNA was collected from 100 mg of protonema tissue, five-days post homogenisation using the RNeasy Plant Mini Kit with on-column DNase I treatment (Qiagen). .. RNA for RNA-seq was sent to GTAC Biotech (Constance, Germany) for library preparation using the TruSeq sample preparation kit (Illumina) and sequencing on the Illumina HiSeq2000 to generate single-end 100 nt long reads.

Article Title: Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease
Article Snippet: .. qRT-PCR RNA was prepared with the RNeasy Mini Kit (74104, QIAGEN). qRT-PCR was performed using pre-designed TaqMan gene expression assays (Thermo Fisher Scientific) and probes values were normalized to an Actb endogenous control. .. Human PMD Cultures Human PMD patient (PLP1 c.254T>G ) and gene-corrected (isogenic control) iPSCs were previously generated as described ( ) and used to generate oligocortical spheroids ( ).

Microarray:

Article Title: Dietary fibers inhibit obesity in mice, but host responses in the cecum and liver appear unrelated to fiber-specific changes in cecal bacterial taxonomic composition
Article Snippet: .. Confirmation of microarray identified differences in gene expression using custom designed RT Profiler PCR arrays Genes showing altered responses to HFD + inulin or HFD + inul PB esters in cecum were identified from microarray analysis and validated using a custom designed RT Profiler PCR Array (Qiagen) (Supplementary File ). .. Real-time PCR Complementary cDNA templates for real-time PCR assays were prepared from Superscript II (Invitrogen) reverse transcribed total RNA and Taqman assays were conducted with duplex target and reference gene UBE2D2 . (Supplementary File ).

Expressing:

Article Title: Dietary fibers inhibit obesity in mice, but host responses in the cecum and liver appear unrelated to fiber-specific changes in cecal bacterial taxonomic composition
Article Snippet: .. Confirmation of microarray identified differences in gene expression using custom designed RT Profiler PCR arrays Genes showing altered responses to HFD + inulin or HFD + inul PB esters in cecum were identified from microarray analysis and validated using a custom designed RT Profiler PCR Array (Qiagen) (Supplementary File ). .. Real-time PCR Complementary cDNA templates for real-time PCR assays were prepared from Superscript II (Invitrogen) reverse transcribed total RNA and Taqman assays were conducted with duplex target and reference gene UBE2D2 . (Supplementary File ).

Article Title: Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease
Article Snippet: .. qRT-PCR RNA was prepared with the RNeasy Mini Kit (74104, QIAGEN). qRT-PCR was performed using pre-designed TaqMan gene expression assays (Thermo Fisher Scientific) and probes values were normalized to an Actb endogenous control. .. Human PMD Cultures Human PMD patient (PLP1 c.254T>G ) and gene-corrected (isogenic control) iPSCs were previously generated as described ( ) and used to generate oligocortical spheroids ( ).

Polymerase Chain Reaction:

Article Title: Dietary fibers inhibit obesity in mice, but host responses in the cecum and liver appear unrelated to fiber-specific changes in cecal bacterial taxonomic composition
Article Snippet: .. Confirmation of microarray identified differences in gene expression using custom designed RT Profiler PCR arrays Genes showing altered responses to HFD + inulin or HFD + inul PB esters in cecum were identified from microarray analysis and validated using a custom designed RT Profiler PCR Array (Qiagen) (Supplementary File ). .. Real-time PCR Complementary cDNA templates for real-time PCR assays were prepared from Superscript II (Invitrogen) reverse transcribed total RNA and Taqman assays were conducted with duplex target and reference gene UBE2D2 . (Supplementary File ).

Derivative Assay:

Article Title: BAK/BAX-Mediated Apoptosis Is a Myc-Induced Roadblock to Reprogramming
Article Snippet: .. Genomic DNA was isolated from six clones of each genotype, plus the corresponding MEFs from which the clones were derived, using a DNeasy blood and tissue kit (Qiagen). .. Libraries were prepared from sheared genomic DNA (∼300 bp; 4 ng per sample) using a Truseq DNA sample preparation kit (Illumina).

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    Qiagen quantitative real time polymerase chain reaction qrt pcr rneasy mini kits
    A. Western blotting analysis showed protein expression of PDCD6 was higher in HO-8910 PM cells than that in HO-8910 cells . GAPDH was used as a loading control. B. Western blotting analysis showed protein expression of PDCD6 in shRNA-PDCD6-HO-8910 PM was decreased compared with that in control HO-8910 PM-mock. GAPDH was used as a loading control. C. <t>qRT-PCR</t> results showed mRNA expression of PDCD6 in shRNA-PDCD6-HO-8910 PM was inhibited by 80% compared with that in HO-8910 PM-mock.
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    A. Western blotting analysis showed protein expression of PDCD6 was higher in HO-8910 PM cells than that in HO-8910 cells . GAPDH was used as a loading control. B. Western blotting analysis showed protein expression of PDCD6 in shRNA-PDCD6-HO-8910 PM was decreased compared with that in control HO-8910 PM-mock. GAPDH was used as a loading control. C. qRT-PCR results showed mRNA expression of PDCD6 in shRNA-PDCD6-HO-8910 PM was inhibited by 80% compared with that in HO-8910 PM-mock.

    Journal: Journal of Translational Medicine

    Article Title: PDCD6 is an independent predictor of progression free survival in epithelial ovarian cancer

    doi: 10.1186/1479-5876-10-31

    Figure Lengend Snippet: A. Western blotting analysis showed protein expression of PDCD6 was higher in HO-8910 PM cells than that in HO-8910 cells . GAPDH was used as a loading control. B. Western blotting analysis showed protein expression of PDCD6 in shRNA-PDCD6-HO-8910 PM was decreased compared with that in control HO-8910 PM-mock. GAPDH was used as a loading control. C. qRT-PCR results showed mRNA expression of PDCD6 in shRNA-PDCD6-HO-8910 PM was inhibited by 80% compared with that in HO-8910 PM-mock.

    Article Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) RNeasy Mini Kits (QIAGEN Inc., Valencia, CA) were used to extract total RNA from cells and tissue.

    Techniques: Western Blot, Expressing, shRNA, Quantitative RT-PCR

    (a) Gel electrophoresis (polyacrylamide 15% stained with ethidium bromide) of LMW RNA samples (LCL) extracted with TRIzol reagent (lane 1), RNEasy kit (lane 2), and small RNA fraction enriched with MirVana kit (lane 3). LMW profile obtained with MirVana kit extraction is similar to that obtained with TRIzol reagent which is not shown for clarity. (b) Agilent 2100 Bioanalyzer electropherogram profile of LMW RNAs (LCL) extracted with TRIzol (black) superimposed on 5.8S (red), 5S (green), and tRNA (blue) bands eluted from polyacrylamide gel. (c) Lymphoblastoid (LCL) LMW RNA profile obtained after plotting the exported raw data from Agilent electropherogram ( ) together with the PeakFit fitted curve (solid line) and the component peak functions. Seven peaks below the LMW RNA profile were fitted by the software ( r 2 = 0.998693).

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Quantification of Small Non-Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles

    doi: 10.1155/2009/659028

    Figure Lengend Snippet: (a) Gel electrophoresis (polyacrylamide 15% stained with ethidium bromide) of LMW RNA samples (LCL) extracted with TRIzol reagent (lane 1), RNEasy kit (lane 2), and small RNA fraction enriched with MirVana kit (lane 3). LMW profile obtained with MirVana kit extraction is similar to that obtained with TRIzol reagent which is not shown for clarity. (b) Agilent 2100 Bioanalyzer electropherogram profile of LMW RNAs (LCL) extracted with TRIzol (black) superimposed on 5.8S (red), 5S (green), and tRNA (blue) bands eluted from polyacrylamide gel. (c) Lymphoblastoid (LCL) LMW RNA profile obtained after plotting the exported raw data from Agilent electropherogram ( ) together with the PeakFit fitted curve (solid line) and the component peak functions. Seven peaks below the LMW RNA profile were fitted by the software ( r 2 = 0.998693).

    Article Snippet: Total RNA Extraction and Small RNAs Enrichment Protocols Total RNA was extracted using three different methods: an acid phenol/guanidine isothiocyanate solution (TRIzol Reagent, Invitrogen), a glass-fiber filtration protocol (MirVana miRNA Isolation Kit, Ambion) that provides also a procedure to isolate and enrich low molecular weight (LMW) RNAs from higher molecular weight (HMW) RNAs, and another common glass-fiber purification protocol (RNEasy Mini Kit, Qiagen).

    Techniques: Nucleic Acid Electrophoresis, Staining, Software

    si RNA knockdown of TRPM 7 channel abrogates Ca 2+ influx and I spont in K562 cells. (A) detection of TRPM 7 transcripts (398 bp) by conventional RT ‐ PCR . Ten μ mol/L doxycycline ( DXC ) almost completely eliminated TRPM 7 mRNA expression. (B) immunoblots of K562 protein extracts by TRPM 7 antibody. Doxycycline concentration dependently (0.1–10 μ mol/L) reduced TRPM 7 protein expression. The results shown in A and B are representative of three independent experiments. (C) fura‐2 Ca 2+ fluorescence imaging from untreated ([ DXC (−)] and 10 μ mol/L doxycycline‐treated [ DXC (+)] K562 cells stably expressing tet ‐inducible TRPM 7‐specific si RNA (si TRPM 7). n = 36 and 26, respectively. (D) density of I spont recorded by conventional whole‐cell recording from untreated ([ DXC (−)] and 10 μ mol/L doxycycline‐treated [ DXC (+)] K562 cells stably expressing si TRPM 7. To facilitate the induction of TRPM 7 currents, ATP and Mg 2+ were absent in the patch pipette. The numbers of cells tested are shown in parentheses.

    Journal: Physiological Reports

    Article Title: TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562. TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562

    doi: 10.14814/phy2.13796

    Figure Lengend Snippet: si RNA knockdown of TRPM 7 channel abrogates Ca 2+ influx and I spont in K562 cells. (A) detection of TRPM 7 transcripts (398 bp) by conventional RT ‐ PCR . Ten μ mol/L doxycycline ( DXC ) almost completely eliminated TRPM 7 mRNA expression. (B) immunoblots of K562 protein extracts by TRPM 7 antibody. Doxycycline concentration dependently (0.1–10 μ mol/L) reduced TRPM 7 protein expression. The results shown in A and B are representative of three independent experiments. (C) fura‐2 Ca 2+ fluorescence imaging from untreated ([ DXC (−)] and 10 μ mol/L doxycycline‐treated [ DXC (+)] K562 cells stably expressing tet ‐inducible TRPM 7‐specific si RNA (si TRPM 7). n = 36 and 26, respectively. (D) density of I spont recorded by conventional whole‐cell recording from untreated ([ DXC (−)] and 10 μ mol/L doxycycline‐treated [ DXC (+)] K562 cells stably expressing si TRPM 7. To facilitate the induction of TRPM 7 currents, ATP and Mg 2+ were absent in the patch pipette. The numbers of cells tested are shown in parentheses.

    Article Snippet: Reverse transcription polymerase reaction (RT‐PCR) and real‐time PCR Total RNA (about 1 μ g) was extracted from about a million of K562 cells with the total RNA extraction kit (RNeasy Minikit, Qiagen) and reverse transcribed (in a 10 μ L scale) by SuperScriptII (Thermo Fisher Scientific) with a randomized or an oligo‐dT primer to obtain the first strand DNA.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Concentration Assay, Fluorescence, Imaging, Stable Transfection, Transferring

    K562 cell growth is dependent on Ca 2+ influx mediated by TRPM 7 channel. (A) Dependency of K562 cell growth on extracellular Ca 2+ concentration evaluated at 72 h after culture. Open and filled circles indicate K562 cells untreated [ DXC (‐)] or treated with 10 μ mol/L doxycycline [ DXC (+)], respectively. (B) time courses of K562 cell growth under various conditions, i.e. DXC (−) (no doxycycline and normal Ca 2+ in RPMI medium), DXC (−)0Ca 2+ (no doxycycline and Ca 2+ free in the medium), DXC (+) (10 μ mol/L doxycycline and normal Ca 2+ in the medium) and DXC (+)0Ca 2+ (10 μ mol/L doxycycline and Ca 2+ free in the medium). In both types of experiments (A and B), K562 cells stably expressing tet ‐inducible TRPM 7‐si RNA (si TRPM 7) were used. n = 5 for each condition. * P

    Journal: Physiological Reports

    Article Title: TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562. TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562

    doi: 10.14814/phy2.13796

    Figure Lengend Snippet: K562 cell growth is dependent on Ca 2+ influx mediated by TRPM 7 channel. (A) Dependency of K562 cell growth on extracellular Ca 2+ concentration evaluated at 72 h after culture. Open and filled circles indicate K562 cells untreated [ DXC (‐)] or treated with 10 μ mol/L doxycycline [ DXC (+)], respectively. (B) time courses of K562 cell growth under various conditions, i.e. DXC (−) (no doxycycline and normal Ca 2+ in RPMI medium), DXC (−)0Ca 2+ (no doxycycline and Ca 2+ free in the medium), DXC (+) (10 μ mol/L doxycycline and normal Ca 2+ in the medium) and DXC (+)0Ca 2+ (10 μ mol/L doxycycline and Ca 2+ free in the medium). In both types of experiments (A and B), K562 cells stably expressing tet ‐inducible TRPM 7‐si RNA (si TRPM 7) were used. n = 5 for each condition. * P

    Article Snippet: Reverse transcription polymerase reaction (RT‐PCR) and real‐time PCR Total RNA (about 1 μ g) was extracted from about a million of K562 cells with the total RNA extraction kit (RNeasy Minikit, Qiagen) and reverse transcribed (in a 10 μ L scale) by SuperScriptII (Thermo Fisher Scientific) with a randomized or an oligo‐dT primer to obtain the first strand DNA.

    Techniques: Concentration Assay, Stable Transfection, Expressing

    Erythroid differentiation of K562 cell by hemin is regulated by Ca 2+ influx mediated by TRPM 7. (A) representative RT ‐ PCR experiments showing hemin‐induced γ ‐globin synthesis in K562 cells untreated (control) and treated with 10 μ mol/L FTY 720 (+ FTY ), 1 mmol/L EGTA (+ EGTA ) or 10 μ mol/L doxycycline (+dxc). In all conditions, K562 cells stably expressing tet ‐inducible TRPM 7‐si RNA (si TRPM 7) were used. The expression of γ ‐globin mRNA was greatly decreased by these procedues, while that of GAPDH stayed almost constant. (B and C) statistical evaluation of pooled data from experiments as shown in A: 10 μ mol/L FTY 720 and 1 mmol/L EGTA (B): si TRPM 7 (C). (D and E) effects of 10 μ mol/L FTY 720 ( FTY ), 1 mmol/L EGTA ( EGTA ) or 10 μ mol/L doxycycline (+dxc) on hemoglobin synthesis at rest or 3 days after hemin treatment in K562‐cells stably expressing si TRPM 7. n = 5 for each condition. * P

    Journal: Physiological Reports

    Article Title: TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562. TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562

    doi: 10.14814/phy2.13796

    Figure Lengend Snippet: Erythroid differentiation of K562 cell by hemin is regulated by Ca 2+ influx mediated by TRPM 7. (A) representative RT ‐ PCR experiments showing hemin‐induced γ ‐globin synthesis in K562 cells untreated (control) and treated with 10 μ mol/L FTY 720 (+ FTY ), 1 mmol/L EGTA (+ EGTA ) or 10 μ mol/L doxycycline (+dxc). In all conditions, K562 cells stably expressing tet ‐inducible TRPM 7‐si RNA (si TRPM 7) were used. The expression of γ ‐globin mRNA was greatly decreased by these procedues, while that of GAPDH stayed almost constant. (B and C) statistical evaluation of pooled data from experiments as shown in A: 10 μ mol/L FTY 720 and 1 mmol/L EGTA (B): si TRPM 7 (C). (D and E) effects of 10 μ mol/L FTY 720 ( FTY ), 1 mmol/L EGTA ( EGTA ) or 10 μ mol/L doxycycline (+dxc) on hemoglobin synthesis at rest or 3 days after hemin treatment in K562‐cells stably expressing si TRPM 7. n = 5 for each condition. * P

    Article Snippet: Reverse transcription polymerase reaction (RT‐PCR) and real‐time PCR Total RNA (about 1 μ g) was extracted from about a million of K562 cells with the total RNA extraction kit (RNeasy Minikit, Qiagen) and reverse transcribed (in a 10 μ L scale) by SuperScriptII (Thermo Fisher Scientific) with a randomized or an oligo‐dT primer to obtain the first strand DNA.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Expressing

    The GATA4-expressing EBs eventually are highly enhanced for liver markers The EBs derived entirely from iGATA4ES cells were induced to express Gata4 at day 2, plated at day 10 under conditions that normally support mesoderm development, and harvested at day 17. Shown are representative qPCR results analyzing cDNA isolated from induced EBs compared with levels of transcripts (as indicated) in the uninduced control cells. The relative levels are plotted with the data log2 transformed. The GATA4-expressing cells have relatively high levels of transcripts for liver markers Afp and Alb , and relatively unchanged levels for the pancreas markers Pdx1 and Ins . Multiple independent experiments gave equivalent results.

    Journal: Developmental biology

    Article Title: Gata4 directs development of cardiac-inducing endoderm from ES cells

    doi: 10.1016/j.ydbio.2009.10.003

    Figure Lengend Snippet: The GATA4-expressing EBs eventually are highly enhanced for liver markers The EBs derived entirely from iGATA4ES cells were induced to express Gata4 at day 2, plated at day 10 under conditions that normally support mesoderm development, and harvested at day 17. Shown are representative qPCR results analyzing cDNA isolated from induced EBs compared with levels of transcripts (as indicated) in the uninduced control cells. The relative levels are plotted with the data log2 transformed. The GATA4-expressing cells have relatively high levels of transcripts for liver markers Afp and Alb , and relatively unchanged levels for the pancreas markers Pdx1 and Ins . Multiple independent experiments gave equivalent results.

    Article Snippet: Total RNA was isolated from EBs (Qiagen RNeasy mini kit). cDNA synthesis was performed using the Transcriptor kit (Roche) and qPCR completed on a Lightcycler 480 II (Roche) using the Lightcycler 480 Sybr Green Kit (Roche).

    Techniques: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Isolation, Transformation Assay

    In EBs that are enhanced for cardiogenesis, the Gata4 -expressing cells commit to endoderm, not mesoderm Shown are representative qPCR results using cDNA derived from cells sorted out of mixed EBs (group 2, 50% iGATA4ES / 50% AinV). EBs were induced with doxycycline at day 2, and at day 3 the GATA4-expressing cells were sorted as GFP+ and compared to levels of transcripts (as indicated) in the GFP−. The normalized expression data is shown log2 transformed. The GATA4− expressing GFP+ cells express relatively high levels of endoderm markers Sox17 and Dkk1 , and relatively low levels for the mesoderm markers. Multiple independent experiments gave similar results.

    Journal: Developmental biology

    Article Title: Gata4 directs development of cardiac-inducing endoderm from ES cells

    doi: 10.1016/j.ydbio.2009.10.003

    Figure Lengend Snippet: In EBs that are enhanced for cardiogenesis, the Gata4 -expressing cells commit to endoderm, not mesoderm Shown are representative qPCR results using cDNA derived from cells sorted out of mixed EBs (group 2, 50% iGATA4ES / 50% AinV). EBs were induced with doxycycline at day 2, and at day 3 the GATA4-expressing cells were sorted as GFP+ and compared to levels of transcripts (as indicated) in the GFP−. The normalized expression data is shown log2 transformed. The GATA4− expressing GFP+ cells express relatively high levels of endoderm markers Sox17 and Dkk1 , and relatively low levels for the mesoderm markers. Multiple independent experiments gave similar results.

    Article Snippet: Total RNA was isolated from EBs (Qiagen RNeasy mini kit). cDNA synthesis was performed using the Transcriptor kit (Roche) and qPCR completed on a Lightcycler 480 II (Roche) using the Lightcycler 480 Sybr Green Kit (Roche).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Transformation Assay