Journal: International Journal of Molecular Sciences

Article Title: The Transcriptional Regulator TfmR Directly Regulates Two Pathogenic Pathways in Xanthomonas oryzae pv. oryzicola

doi: 10.3390/ijms25115887

Figure Lengend Snippet: Constitutive expression of rpfG restores motility and EPS production of the mutant Δ tfmR . And Xoc TfmR binds directly to the promoter of RpfG and activates its transcription. ( A ) Constitutive expression of rpfG restores motility of the mutant Δ tfmR . ( i ) Example photo of a bacterial strain. ( ii ) Mean measurements of colony diameter for each strain on “swarming” plates. ( iii ) Mean measurements of colony diameter for each strain on “swimming” plates. Data shown are the mean ± SD ( n = 10). Significance was determined by ANOVA and Dunnett’s post hoc test for comparison with to the wild type. ** p < 0.01; n.s., not significant. ( B ) Constitutive expression of rpfG restores the yield of the mutant Δ tfmR EPS. ( i ) Xoc strains were grown on NA plates supplemented with 2% sucrose for 3 days. ( ii ) Xoc strains were cultured in NB medium supplemented with 2% sucrose for 3 days and EPS was precipitated from the culture supernatant. Values given are the means ± SD of triplicate measurements from a representative experiment, and significance was determined by analysis of variance (ANOVA) and Dunnett’s post hoc test for comparison with the wild type. * p < 0.05; n.s., not significant. Similar results were obtained in two other independent experiments. ( C ) Electrophoretic mobility shift and competition assays of TfmR with the promoter region of rpfG ( i ) and hutG ( ii ) (negative control); the bound– and free–DNA fragments are marked with the words Bound probe and Free probe, respectively, and the concentrations are indicated at the top of each lane. ( D ) ß–Glucuronidase (GUS) activity of the gusA reporter of the rpfG gene promoter in the Δ tfmR mutant and the wild type in NB medium ( i ), or in XOM2 medium ( ii ). The data shown are the mean and standard deviation of three measurements. The experiment was repeated three times and similar results were obtained. Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( E ) Detection of Δ tfmR mutant and wild–type expression of rpf genes in NB medium ( i ), or XOM2 medium ( ii ), revealed by RT–qPCR analysis. Values are the means ± SD ( n = 3 biological replicates). Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( F ) Fold enrichment of the promoter region of rpfG in the GX01/TfmR::3 × Flag–ChIP samples compared with the Mock–ChIP samples (with anti–HA antibody), as measured by ChIP–qPCR using hutG as the negative control. Data are presented as means ± SD ( n = 3). Differences were evaluated using Student’s t -test (* p < 0.05; n.s., no significance at p ≤ 0.05). ( G ) In vitro transcription experiments showing TfmR activates the transcription of rpfG . RNA was produced from a 323 bp template DNA fragment containing the rpfG promoter using E. coli RNA polymerase (RNAP) holoenzyme. A 334 bp template DNA fragment containing the hutG promoter and a 150 bp template DNA fragment of the rpfG coding sequence were used as controls. Lane 1, template DNA alone; lane 2, template DNA with RANP; lanes 3–4, template DNA with RANP and 5 and 10 nM TrxA–TfmR.

Article Snippet: The TrxA-TfmR protein and DNA fragments were incubated in transcription buffer at 28 °C for 30 min. Then, the NTP mixture (250 μM each of ATP, CTP, and GTP; 250 μM biotin 16-UTP) and 0.5 U of E. coli RNA polymerase holoenzyme (New England BioLabs, Ipswich, MA, USA) were added and the reaction was carried out for 1 h at 37 °C.

Techniques: Expressing, Mutagenesis, Comparison, Cell Culture, Electrophoretic Mobility Shift Assay, Negative Control, Activity Assay, Standard Deviation, Quantitative RT-PCR, In Vitro, Produced, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: The Transcriptional Regulator TfmR Directly Regulates Two Pathogenic Pathways in Xanthomonas oryzae pv. oryzicola

doi: 10.3390/ijms25115887

Figure Lengend Snippet: Xoc TfmR binds directly to the promoter of HrpX and activates its transcription. ( A ) ß–Glucuronidase (GUS) activity of the gusA reporter of the hrpG and hrpX gene promoter in the Δ tfmR mutant and the wild type in NB medium ( i ), or in XOM2 medium ( ii ). The data shown are the mean and standard deviation of three measurements. The experiment was repeated three times and similar results were obtained. Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( B ) Detection of Δ tfmR –mutant and wild-type expression of T3SS genes in NB medium ( i ), or XOM2 medium ( ii ) revealed by RT–qPCR analysis. Values are the means ± SD ( n = 3 biological replicates). Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( C ) Electrophoretic mobility shift and competition assays of TfmR with the promoter region of hrpX ( i ) and hutG ( ii ) (negative control); the bound– and free–DNA fragments are marked with the words Bound probe and Free probe, respectively, and the concentrations are indicated at the top of each lane. ( D ) Fold enrichment of the promoter region of hrpX in the GX01/TfmR::3 × Flag–ChIP samples compared with the Mock–ChIP samples (with anti–HA antibody), as measured by ChIP–qPCR using hutG as the negative control. Data are presented as means ± SD ( n = 3). Differences were evaluated using Student’s t –test (* p < 0.05; n.s., no significance at p ≤ 0.05). ( E ) In vitro transcription experiments showing TfmR activates the transcription of hrpX . RNA was produced from a 371 bp template DNA fragment containing the hrpX promoter using E. coli RNA polymerase (RNAP) holoenzyme. A 334 bp template DNA fragment containing the hutG promoter and a 161 bp template DNA fragment of the hrpX coding sequence were used as controls. Lane 1, template DNA alone; lane 2, template DNA with RANP; lanes 3–4, template DNA with RANP and 5 and 10 nM TrxA–TfmR.

Article Snippet: The TrxA-TfmR protein and DNA fragments were incubated in transcription buffer at 28 °C for 30 min. Then, the NTP mixture (250 μM each of ATP, CTP, and GTP; 250 μM biotin 16-UTP) and 0.5 U of E. coli RNA polymerase holoenzyme (New England BioLabs, Ipswich, MA, USA) were added and the reaction was carried out for 1 h at 37 °C.

Techniques: Activity Assay, Mutagenesis, Standard Deviation, Expressing, Quantitative RT-PCR, Electrophoretic Mobility Shift Assay, Negative Control, In Vitro, Produced, Sequencing

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: Enrichment of C‐type lectin domain family 4 member E (CLEC4E) in tumour‐associated macrophage (TAM) is correlated with unfavourable patient prognosis. (A) Volcano plot of gene enrichment in TAMs by RNA sequencing. (B) Heatmap of gene expressions in M0 and TAM by RNA sequencing, ranked by expression level in TAM. (C) Immunofluorescence of paired tumour and tumour adjacent tissues from melanoma patients. (D) Comparison of CLEC4E fluorescence intensity and macrophage count per field between paired tumour and tumour adjacent tissues. (E) CLEC4E fluorescence comparison between tumours from melanoma patients in stage I/II versus stage III/IV. (F) Overall survival analysis of patients with CLEC4E high and low expressions. Median CLEC4E fluorescence level was determined as the cutoff. (G) Overall survival analysis of patients with high CD68 + infiltration and high CLEC4E expression and patients with low CD68 + infiltration and low CLEC4E expression. Median CLEC4E fluorescence level and median CD68 + infiltration level were determined as the cutoffs.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: RNA Sequencing, Expressing, Immunofluorescence, Comparison, Fluorescence

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: C‐type lectin domain family 4 member E (CLEC4E) expression on tumour‐associated macrophage (TAM) promotes tumour growth in mouse models. (A) Workflow of melanoma mouse model of CLEC4E conditional knockout mice. (B) Tumour growth curve of B16F10 melanoma model. (C) Survival analysis of B16F10 melanoma model. Tumour volume exceeding 500 mm 3 was considered as the endpoint. (D) Pictures of melanoma tissues from CLEC4E knockout and control groups. (E) Body weight gain since intraperitoneal injection of ID8 cells in ovarian cancer model. (F) Survival analysis of ID8 ovarian cancer model. Body weight gain exceeding 4 g was considered as the endpoint. (G) Comparison of celiac tumour implantations in CLEC4E knockout and control groups. (H) Representative pictures of intestinal implantations. (I) Flow cytometry analysis of CD206 and CD68 from melanoma tissues at day 10. (J) Flow cytometry analysis of CD206 and CD68 from ovarian model ascites at week 8.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: Expressing, Knock-Out, Control, Injection, Comparison, Flow Cytometry

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: Single‐cell RNA sequencing analysis of macrophages from melanoma tissues of C‐type lectin domain family 4 member E (CLEC4E) knockout and control mice. (A) Uniform manifold approximation and projection (UMAP) plot of total macrophages. (B) Bar chart showing proportions of macrophage clusters in CLEC4E knockout and control mice. (C) Pseudotime trajectory of macrophages. Cells were divided into five states. (D) Pseudotime trajectories and bar chart showing macrophage distributions of CLEC4E knockout and control mice. (E) Heatmap showing gene markers of each state and the proportion comparison between knockout and control mice. CLEC4E knockout mice had enriched macrophages in states 1 and 4, and decreased macrophages in states 2 and 5 compared to control mice.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: RNA Sequencing, Knock-Out, Control, Comparison

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: C‐type lectin domain family 4 member E (CLEC4E) deletion suppresses tumour‐associated macrophage (TAM) proliferation and abundance in tumour microenvironment (TME). (A) Immunofluorescence of CD68 and Ki67 with B16F10 melanoma tumours from CLEC4E knockout and control mice. (B) Column charts comparing Ki67 + cells in TAM and macrophage count between control and CLEC4E knockout groups. (C) Macrophage sorting chart and qRT‐PCR comparing proliferation markers in macrophages from control and CLEC4E knockout mice. (D) qRT‐PCR of CLEC4E silencing efficiency and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis with CLEC4E silencing in TAM. (E) RT‐PCR verification of proliferation gene markers enriched in KEGG analysis. (F) Top 10 differentially expressed phosphorylated proteins between CLEC4E knockout and control macrophages. (G) Protein‒protein interaction (PPI) analysis of differentially expressed phosphorylated proteins in CLEC4E knockout versus control macrophages. (H) Western blot of PLC‐γ2, Syk and Erk phosphorylation with CLEC4E ligation in macrophages. (Trehalose‐6,6‐dibehenate) TDB concentrations were 0, 10, 25 and 50 µg/mL sequentially. (I) Cell counting kit‐8 assay of M0 and TAMs differentiated from RAW264.7 with TDB or Erk inhibitor (Erki). (j) Cell counting kit‐8 assay of peritoneal macrophages from CLEC4E knockout and control mice.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: Immunofluorescence, Knock-Out, Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Ligation, Cell Counting

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: C‐type lectin domain family 4 member E (CLEC4E) knockout strengthens macrophage‒T cell interaction and T‐cell cytotoxicity. (A) Interactions of all cell clusters in control and CLEC4E knockout mice. (B) Bar charts showing the number and strength of total interactions. (C) Bar chart of interaction numbers of tumour‐associated macrophage (TAM)‒T cells. (D) Selective ligand‒receptor pair expressions between TAM and T cells. (E) Bar chart of RT‐PCR with sorted macrophages from mouse melanoma tissues. (F) Expressions of selective genes in T‐cell population. (G) Flow cytometry of granzyme B and CD8 in ovarian cancer ascites at week 18. (H) Flow cytometry of CD4 and CD8 in melanoma tissues at day 10. (I) Immunohistochemistry of granzyme B with intestinal implantation of ovarian cancer. (J) Immunofluorescence images of melanoma tumour tissues from two patients and the correlation between the area of mean fluorescence of CLEC4E and CD8 of 18 patients.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: Knock-Out, Control, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Immunohistochemistry, Immunofluorescence, Fluorescence

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: BET inhibitor strongly suppresses C‐type lectin domain family 4 member E (CLEC4E) expression on tumour‐associated macrophage (TAM). (A) Screening of 132 drugs for CLEC4E inhibition in RAW264.7 TAM induced by B16‐CM. (B) RT‐PCR of CLEC4E expression on BMDM with B16‐CM and NHWD‐870 treatment. (C) Western blot of CLEC4E expression on BMDM with B16‐CM and NHWD‐870 treatment. (D) Western blot of CLEC4E expression on THP‐1 TAM induced with SK28‐CM or A2780‐CM. (E) Immunofluorescence of Yumm1.7 melanoma tissues with BET inhibitor NHWD‐870 treatment.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: Expressing, Inhibition, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: Targeting CLEC4E in immunosuppressive tumour‐associated macrophages via BET inhibition

doi: 10.1002/ctm2.70505

Figure Lengend Snippet: BET inhibitor downregulates C‐type lectin domain family 4 member E (CLEC4E) by targeting on BRD4/CEBPβ. (A) RT‐PCR showing the efficiency of RNA silencing of BRD2, BRD3 and BRD4 on BMDM‐TAM. (B) Western blot of CLEC4E with BRD2/3/4 silenced in BMDM‐TAM. (C) CHIP sequencing (CHIP‐seq) of A375 showing no BRD4 binding on CLEC4E. (D) Western blot of CLEC4E with CEBPβ silenced in BMDM‐TAM. (E) Western blot of CEBPβ with BRD2/3/4 silenced in BMDM‐TAM. (F) Western blot of CEBPβ with BRD4 silenced in THP‐1 tumour‐associated macrophage (TAM). (G) Western blot of CEBPβ with BET inhibitor NHWD‐870 (20 nM) treatment in THP‐1 TAM induced by indicated tumour conditioned medium. (H) Luciferase assay with 293T cells transfected with pGL3‐CEBPB or pLG3‐basic and BRD4 or NC plasmids. (I) CHIP‐seq of A375 showing BRD4 binding on the promoter of CEBPB.

Article Snippet: Wild‐type C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. Clec4e flox/+ (C57BL/6J‐Clec4e em1cyagen ) and Lyz2‐cre mice were purchased from Cyagen Biosciences.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, ChIP-sequencing, Binding Assay, Luciferase, Transfection

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: The Egr-1 gene is rapidly induced after TCR-mediated activation of the DPK double positive cell line. ( A ) Total RNA isolated from DPK cells cultured with immobilized anti-CD3ε mAb for the indicated times (shown in hours), was subjected to RT-PCR analysis using Egr-1 or CD4 primers. ( B ) Electrophoretic mobility shift assay using nuclear lysates prepared from DPK cells 8 h after activation by immobilized antiCD3ε mAb. Probes contained a single Egr-1 binding site ( left ) or overlapping Egr-1 and Sp1 sites ( right ). ( C ) DPK cells were cultured with DCEK-ICAM fibroblast antigen presenting cells and 1 μm pigeon cytochrome c peptide for the indicated times. Total RNA was isolated and subjected to a competitive RT-PCR assay (see Materials and Methods). Note the different scales for Egr-1 and CD4 mRNA expression.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Activation Assay, Isolation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Electrophoretic Mobility Shift Assay, Binding Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: DPK cell differentiation and Egr-2,3 mRNA induction is cyclosporin A sensitive, while Egr-1 mRNA induction is cyclosporin A resistant. ( A , B ) DPK cells were cultured with DCEK-ICAM fibroblast antigen presenting cells and 2 μM pigeon cytochrome c peptide in the presence or absence of 100 ng/ml cyclosporin A, or appropriate dilution of solvent (DMSO) as indicated. Cells were harvested and stained for CD69 after 1 or 3 d in culture ( A ) or stained for CD4 and CD8 after 3 d in culture ( B ). ( C ) RT-PCR analysis of total RNA derived from DPK cells activated for 6 h with immobilized anti-CD3ε mAb in the presence or absence of 300 ng/ml cyclosporin A, using Egr-1, Egr-2 (Krox-20), CD4, CD69 or Egr-3 primers. (*) Also shown for the indicated samples is the relative level of Egr-1 cDNA normalized to expression of CD4 cDNA as determined by competitive RT-PCR assay. The identity of the lower major band in Egr-3 RT-PCR was verified by sequencing.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Cell Differentiation, Cell Culture, Staining, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Expressing, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr gene family is dependent upon ras signaling pathways. ( A ) Competitive RT-PCR was used to compare expression of Egr-1 and CD4 genes in DPK or 17N4 cells activated for 6 h with immobilized anti-CD3ε mAb. ( B ) RT-PCR analysis of total RNA derived from DPK or 17N4 cells activated for 6 h with immobilized anti-CD3ε mAb. Independent PCR reactions using Egr-2 (Krox-20), CD4, or Egr-3 primers were performed.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Egr-1 mRNA and DNA binding activity in the thymus is MHC dependent. ( A ) Thymocytes derived from wild-type or MHCdeficient mice were two color-stained for CD4 and CD8. ( B ) Competitive RT-PCR was used to determine the level of expression of CD4 and Egr-1 genes in thymocytes derived from wild-type or MHC-deficient mice. ( C ) Electrophoretic mobility shift assay using nuclear lysates prepared from freshly isolated thymocytes derived from wild-type or MHCdeficient mice using a probe containing an Egr-1 binding site. Nuclear extracts derived from 5 × 10 5 cells containing equivalent amounts of protein were used in binding reactions. In some instances as indicated, binding reactions contained anti-Egr-1 antibody or normal rabbit serum (NRS). For comparison, a binding reaction containing recombinant Egr-1 is shown.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Binding Assay, Activity Assay, Derivative Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Electrophoretic Mobility Shift Assay, Isolation, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr-1 mRNA in double positive thymocytes. ( A ) Total thymocytes and CD4 + 8 + thymocytes (isolated by cell sorting, 95% DP) derived from the same animal, were assayed for expression of Egr and CD4 mRNA by competitive RT-PCR. Shown is the relative level of Egr-1 cDNA in the sample, normalized to the level of CD4 cDNA. ( B ) Total thymocytes derived from an MHC-deficient mouse were cultured with hamster immunoglobulin-coated or anti-CD3ε mAbcoated beads for 90 min before determination of Egr-1 and CD4 gene expression as in Fig. C .

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Isolation, FACS, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr-1 protein in the thymus. Thin sections of normal thymus ( A–C ) or MHC knockout thymus ( D ) were fixed in formaldehyde and stained with a specific rabbit anti-Egr-1 peptide antiserum ( A , C , D ) or the same antibody preincubated with specific peptide ( B ). Regions of cortex ( C ) and medulla ( M ) are indicated. Sections were counterstained with hematoxylin and photographed at ×20 ( A , B ) or ×40 ( C , D ). C shows a magnification of the same section photographed in A .

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Knock-Out, Staining

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr-1 protein in thymocyte subsets. Thymocytes from a young adult mouse were 4-color stained for expression of CD4, CD8, Egr-1, and CD69 or CD3, and analyzed by FACS ® as described in Materials and Methods. Indicated in the dot plots are the percentage of thymocytes within each quadrant, or in parenthesis ( upper right dot plot ), the percentage of Egr-1 + thymocytes within each thymocyte subset. Where indicated, staining is shown for gated populations of thymocytes (either Egr-1 + thymocytes as shown in histogram, or CD4 + 8 + thymocytes). Similar results were obtained from three other individual animals.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: The Endoplasmic Reticulum pool of Bcl-xL dampens the Unfolded Protein Response through IP3R-dependent Calcium Release

doi: 10.1101/2021.01.27.428229

Figure Lengend Snippet: (A) Western blot detecting Bcl-xL endogenous levels in WT and bclx KO MEFs. α-Tubulin was used as a loading control. (B) Cell death quantification (% of Sytox GREEN™marked cells) in WT and bclx KO MEFs treated with 1µM Staurosporine for 24hrs or 1µM Thapsigargin for 48 hrs (mean ± SEM; n=3; *, p<0.05; **, P<0.01). Results are displayed at t=15hrs and t=32hrs, respectively. C) Representative images of cleaved Caspase 3 stained WT and KO MEFS after Staurosporine and Thapsigargin treatment for 6hrs and 24hrs, respectively. Blue fluorescence: Nuclei. Red fluorescence: cleaved Caspase 3. Scale bar: 10µm. (D) Quantification of Cleaved Caspase 3 stained WT and KO MEFs after treatment with 250nM Staurosporine for 6hrs or 4µM Thapsigargin for 24hrs. 5 images of an average of 30 cells each were taken per cell type per experiment (n=3). Control: treated with DMSO (mean ± SEM; n=3; *, p<0.05; ***, p<0.001). (E) Kinetics of UPR markers in the presence of 4μM Thapsigargin for 24hrs in WT and bclx KO MEFs. α-Tubulin was used as a loading control.

Article Snippet: The following antibodies were used: Vinculin (Santa Cruz #sc-55465; 1/2000), Purified Mouse Anti-ATP Synthase β – F0F1 (BD Transduction Laboratories #612518; 1/2000), Calnexin (Cell signaling #2679; 1/1000), α-Tubulin (Santa Cruz #sc-32293; 1/1000), Actin (Sigma-Aldrich #A2066; 1/500), PERK (Cell signaling #3192; 1/1000), phospho-PERK (Cell signaling #3179; 1/1000), eIF2α (Cell Signaling #2103, 1/1000), phospho-eIF2α (Cell Signaling #3398, 1/1000), ATF4 (Cell Signaling #11815, 1/1000), CHOP (Cell Signaling #5554, 1/1000), Bcl-xL (Cell Signaling #2764, 1/1000), PARP (abcam #ab6079, 1/500), Cleaved Caspase 3 (Cell Signaling #9661, 1/1000).

Techniques: Western Blot, Control, Staining, Fluorescence

Journal: bioRxiv

Article Title: The Endoplasmic Reticulum pool of Bcl-xL dampens the Unfolded Protein Response through IP3R-dependent Calcium Release

doi: 10.1101/2021.01.27.428229

Figure Lengend Snippet: (A) The mice genotyping strategy was done with a Cre-LoxP recombination system where an engineered vector containing the WT Bcl-xL exon 3 (E3) and CB5 or ActA mutant sequences targeting Bcl-xL either to the ER or mitochondria, respectively, was inserted to replace the WT Bcl-xL E3 in floxed mice. Cre-recombination induced WT exon 3 excision and CB5 or ActA mutant expression. Accordingly, three protein products are possible: WT Bcl-xL with intact TM domain, ER-xL with CB5 targeting sequence and Mt-xL with ActA targeting sequence. Neomycin is used as an inducer of vector resistance (Neo R ). Two primers were used: XTAG-Forward and XTAG-Reverse. Poly(A): Poly(A) tail. (B) Western blot detecting Bcl-xL endogenous levels in cells extracted from the lung, liver and brain of WT, ER-xL and Mt-xL mice. Vinculin and actin were used as loading controls. (C) Western blot detecting Bcl-xL endogenous levels in WT, ER-xL and Mt-xL MEFs extracted at E13. α-tubulin is used as a loading control. (D) Bcl-xL subcellular localization detection in WT, ER-xL and Mt-xL MEFs by Immunofluorescence. Co-localization was assessed using ER-EGFP transfection and Mitotracker staining for ER and mitochondrial localization, respectively. Profile plots of fluorescence signals detecting the intensity of fluorescence along the white segments on merged images were quantified by ImageJ software and shown to the right. Scale bar: 10µm. (F) Bcl-xL endogenous expression in ER-xL (Top panel) and Mt-xL (lower panel) MEFs post-subcellular fractionation was detected by western blot. Vinculin is used as a cytosol marker, Calnexin as an ER marker and F0F1 ATPase as a mitochondrial maker. (Tot) whole-cell lysates; (Mito) mitochondria; (Cyto) cytosol.

Article Snippet: The following antibodies were used: Vinculin (Santa Cruz #sc-55465; 1/2000), Purified Mouse Anti-ATP Synthase β – F0F1 (BD Transduction Laboratories #612518; 1/2000), Calnexin (Cell signaling #2679; 1/1000), α-Tubulin (Santa Cruz #sc-32293; 1/1000), Actin (Sigma-Aldrich #A2066; 1/500), PERK (Cell signaling #3192; 1/1000), phospho-PERK (Cell signaling #3179; 1/1000), eIF2α (Cell Signaling #2103, 1/1000), phospho-eIF2α (Cell Signaling #3398, 1/1000), ATF4 (Cell Signaling #11815, 1/1000), CHOP (Cell Signaling #5554, 1/1000), Bcl-xL (Cell Signaling #2764, 1/1000), PARP (abcam #ab6079, 1/500), Cleaved Caspase 3 (Cell Signaling #9661, 1/1000).

Techniques: Plasmid Preparation, Mutagenesis, Expressing, Sequencing, Western Blot, Control, Immunofluorescence, Transfection, Staining, Fluorescence, Software, Fractionation, Marker

Journal: bioRxiv

Article Title: The Endoplasmic Reticulum pool of Bcl-xL dampens the Unfolded Protein Response through IP3R-dependent Calcium Release

doi: 10.1101/2021.01.27.428229

Figure Lengend Snippet: (A) Quantification of cleaved Caspase 3 stained WT, ER-xL and Mt-xL MEFs after treatment with 250nM Staurosporine for 6 hrs. 5 images of an average of 30 cells each were taken per cell type per experiment (n=3). Control: treated with DMSO (mean ± SEM; n=3; ***, p<0.001; n.s., non-significant, p>0,05). (B) Kinetics of PARP cleavage in WT, ER-xL and Mt-xL MEFs following 1µM Staurosporine treatment over 12 hours. α-Tubulin was used as a loading control. (C) Cell death quantification (% of Sytox GREEN™-positive cells) in WT, ER-xL and Mt-xL MEFs treated with 1µM Thapsigargin or 2µg/mL Tunicamycin for 72 hrs (mean ± SEM; n=3; n.s., non-significant, p>0.05; **, P<0.01). Results are displayed at t=72hrs. (D) Quantification of cleaved Caspase 3 stained WT and KO MEFs after treatment with 4µM thapsigargin for 24hrs. 5 images of an average of 30 cells each were taken per cell type per experiment (n=3). Control: treated with DMSO (mean ± SEM; n=3; n.s., non-significant, p>0.05; ***, p<0.001). (E) Kinetics of PARP cleavage in WT, ER-xL and Mt-xL MEFs following 4µM Thapsigargin treatment over 24 hours. α-Tubulin was used as a loading control.

Article Snippet: The following antibodies were used: Vinculin (Santa Cruz #sc-55465; 1/2000), Purified Mouse Anti-ATP Synthase β – F0F1 (BD Transduction Laboratories #612518; 1/2000), Calnexin (Cell signaling #2679; 1/1000), α-Tubulin (Santa Cruz #sc-32293; 1/1000), Actin (Sigma-Aldrich #A2066; 1/500), PERK (Cell signaling #3192; 1/1000), phospho-PERK (Cell signaling #3179; 1/1000), eIF2α (Cell Signaling #2103, 1/1000), phospho-eIF2α (Cell Signaling #3398, 1/1000), ATF4 (Cell Signaling #11815, 1/1000), CHOP (Cell Signaling #5554, 1/1000), Bcl-xL (Cell Signaling #2764, 1/1000), PARP (abcam #ab6079, 1/500), Cleaved Caspase 3 (Cell Signaling #9661, 1/1000).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: The Endoplasmic Reticulum pool of Bcl-xL dampens the Unfolded Protein Response through IP3R-dependent Calcium Release

doi: 10.1101/2021.01.27.428229

Figure Lengend Snippet: Kinetics of UPR markers in the presence of 2μM ( A ) and 4μM ( B ) Thapsigargin over 24hrs in WT, ER-xL and Mt-xL MEFs. α-tubulin was used as a loading control. ( C ) RT-PCR assay was used to analyze the mRNA levels of the UPR markers xbp1s, chop and atf4 in WT, ER-xL and Mt-xL MEFs after 4μM thapsigargin treatment over 24 hours.

Article Snippet: The following antibodies were used: Vinculin (Santa Cruz #sc-55465; 1/2000), Purified Mouse Anti-ATP Synthase β – F0F1 (BD Transduction Laboratories #612518; 1/2000), Calnexin (Cell signaling #2679; 1/1000), α-Tubulin (Santa Cruz #sc-32293; 1/1000), Actin (Sigma-Aldrich #A2066; 1/500), PERK (Cell signaling #3192; 1/1000), phospho-PERK (Cell signaling #3179; 1/1000), eIF2α (Cell Signaling #2103, 1/1000), phospho-eIF2α (Cell Signaling #3398, 1/1000), ATF4 (Cell Signaling #11815, 1/1000), CHOP (Cell Signaling #5554, 1/1000), Bcl-xL (Cell Signaling #2764, 1/1000), PARP (abcam #ab6079, 1/500), Cleaved Caspase 3 (Cell Signaling #9661, 1/1000).

Techniques: Control, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: The Endoplasmic Reticulum pool of Bcl-xL dampens the Unfolded Protein Response through IP3R-dependent Calcium Release

doi: 10.1101/2021.01.27.428229

Figure Lengend Snippet: (A) Kinetics of UPR markers after 4µM Thapsigargin and 100µM 2-APB treatment over 24 hours in WT, ER-xL and Mt-xL MEFs. 2-APB is considered as an IP3R inhibitor. α-Tubulin was used as a loading control. (B) Kinetics of UPR markers after 4µM Thapsigargin and 1µM Xestospongin C treatment over 24 hours in WT, ER-xL and Mt-xL MEFs. Xestospongin C is an IP3R and SERCA inhibitor. α-Tubulin was used as a loading control. (C) Proposed model for the role of Bcl-xL at the ER. ER stress might deplete ER Ca 2+ stocks resulting in: 1-mitochondrial apoptosis initiation through mPTP opening and 2-the UPR activation leading to the transcription of genes promoting apoptosis. Hence, the outcome is cell death. Under these conditions, Bcl-xL at the ER interacts with and inhibits the IP3R abrogating ER Ca 2+ depletion required for apoptosis induction. Consequently, Bcl-xL at the ER performs a new indirect anti-apoptotic function upon ER stress.

Article Snippet: The following antibodies were used: Vinculin (Santa Cruz #sc-55465; 1/2000), Purified Mouse Anti-ATP Synthase β – F0F1 (BD Transduction Laboratories #612518; 1/2000), Calnexin (Cell signaling #2679; 1/1000), α-Tubulin (Santa Cruz #sc-32293; 1/1000), Actin (Sigma-Aldrich #A2066; 1/500), PERK (Cell signaling #3192; 1/1000), phospho-PERK (Cell signaling #3179; 1/1000), eIF2α (Cell Signaling #2103, 1/1000), phospho-eIF2α (Cell Signaling #3398, 1/1000), ATF4 (Cell Signaling #11815, 1/1000), CHOP (Cell Signaling #5554, 1/1000), Bcl-xL (Cell Signaling #2764, 1/1000), PARP (abcam #ab6079, 1/500), Cleaved Caspase 3 (Cell Signaling #9661, 1/1000).

Techniques: Control, Activation Assay

Journal: Tissue Engineering. Part A

Article Title: Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Bladder Cells: Potential for Urological Tissue Engineering

doi: 10.1089/ten.tea.2009.0625

Figure Lengend Snippet: Sequence Information of Primers Used for Reverse Transcriptase–Polymerase Chain Reaction and Their Expected Product Size

Article Snippet: The protein was transferred to a poly vinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA) and probed with primary antibodies (alpha-smooth muscle actin [α-SMA, 1:5000; Sigma, St. Louis, MO], calponin [1:1000; Santa Cruz, Santa Cruz, CA], desmin [1:500; Santa Cruz], smooth muscle myosin heavy chain [1:1000; Sigma], uroplakin-Ia [Up-Ia, 1:500, a cell marker for superficial urothelial cells 15 ; Santa Cruz], cytokeratin-13 [CK13, 1:500, a cell marker for urothelial base cells 16 , 17 ; Santa Cruz], cytokeratins [AE1/AE3, 1:1000; Dako, Glostrup, Denmark], and β-actin [1:5000; Sigma]) overnight at 4°C with constant shaking.

Techniques: Sequencing

Journal: Tissue Engineering. Part A

Article Title: Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Bladder Cells: Potential for Urological Tissue Engineering

doi: 10.1089/ten.tea.2009.0625

Figure Lengend Snippet: Expression of muscle and urothelial lineage-specific transcripts in differentiated human BMSCs by reverse transcriptase–polymerase chain reaction on day 14. (A) Smooth muscle-specific primers (α-SMA, desmin, calponin, MHC) used on RNA extracted from human BMSCs (p4) induced with human bladder SMC-derived conditional medium for 14 days. Lane 1, fresh smooth muscle tissue; lane 2, cultured SMCs as positive controls; lane 3, cultured urothelium as negative control; lane 4, noninduced BMSCs as control; lane 5, induced BMSCs by coculture with SMCs; lane 6, induced BMSCs by SMC-CM; lane 7, no template control. (B) Urothelial-specific primers (Up-Ia, CK-7/13) used on RNA samples from human BMSCs (p4) induced by coculture with urothelium for 7 days. Lane 1, cultured urothelium as positive; lane 2, noninduced BMSCs as control; lane 3, BMSCs cocultured with urothelium; lane 4, BMSCs cultured using urothelium-CM; lane 5, no template (H2O) control. GAPDH was used as the housekeeping gene for load control. Note: Threefold excess of the reaction mixture was loaded for all the primers in (A) compared with that in (B). Moreover, the bands for all the primers in (A) are overexposed, whereas the bands in (B) are under exposured. α-SMA, alpha smooth muscle actin; MHC, myosin heavy chain; Up-Ia, uroplakin-Ia; CK-7/13, cytokeratin-7/13; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The protein was transferred to a poly vinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA) and probed with primary antibodies (alpha-smooth muscle actin [α-SMA, 1:5000; Sigma, St. Louis, MO], calponin [1:1000; Santa Cruz, Santa Cruz, CA], desmin [1:500; Santa Cruz], smooth muscle myosin heavy chain [1:1000; Sigma], uroplakin-Ia [Up-Ia, 1:500, a cell marker for superficial urothelial cells 15 ; Santa Cruz], cytokeratin-13 [CK13, 1:500, a cell marker for urothelial base cells 16 , 17 ; Santa Cruz], cytokeratins [AE1/AE3, 1:1000; Dako, Glostrup, Denmark], and β-actin [1:5000; Sigma]) overnight at 4°C with constant shaking.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Derivative Assay, Cell Culture, Negative Control, Control

Journal: Tissue Engineering. Part A

Article Title: Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Bladder Cells: Potential for Urological Tissue Engineering

doi: 10.1089/ten.tea.2009.0625

Figure Lengend Snippet: Myogenic differentiation of human BMSCs using SMC-derived CM. Human BMSCs (p4) were stained with α-SMA (a, e, i), calponin (b, f, j), desmin (c, g, k), and myosin (d, h, l) antibodies without induction as negative control (a–d) and with induction for 14 days (e–h). SMCs were also stained with the same antibodies as a positive control (i–l). Scale bar = 50 μM. Color images available online at www.liebertonline.com/ten.

Article Snippet: The protein was transferred to a poly vinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA) and probed with primary antibodies (alpha-smooth muscle actin [α-SMA, 1:5000; Sigma, St. Louis, MO], calponin [1:1000; Santa Cruz, Santa Cruz, CA], desmin [1:500; Santa Cruz], smooth muscle myosin heavy chain [1:1000; Sigma], uroplakin-Ia [Up-Ia, 1:500, a cell marker for superficial urothelial cells 15 ; Santa Cruz], cytokeratin-13 [CK13, 1:500, a cell marker for urothelial base cells 16 , 17 ; Santa Cruz], cytokeratins [AE1/AE3, 1:1000; Dako, Glostrup, Denmark], and β-actin [1:5000; Sigma]) overnight at 4°C with constant shaking.

Techniques: Derivative Assay, Staining, Negative Control, Positive Control

Journal: Aging (Albany NY)

Article Title: Silencing of the small GTPase DIRAS3 induces cellular senescence in human white adipose stromal/progenitor cells

doi: 10.18632/aging.101197

Figure Lengend Snippet: ( A ) Morphology of ASCs infected with shCntrl and shDIRAS3 was documented using light microscope at 40x magnification. (B and C) ASCs infected with either shDIRAS3 or shControl (shCntrl) expressing lentiviruses were fixed and stained for SA-β-GAL. Percentage of SA-β-GAL positive cells was calculated by scanning 5 low-power magnification fields (n=3). (D) ASCs were transduced by indicated lentiviruses with increasing MOI and cell lysates immunoblotted using phospho-Ser-139 Gamma H2A.X antibody. β-Actin served as a loading control. DIRAS3 was KD in ASCs using lentiviruses expressing specific shRNA at increasing MOI. Cell lysates were immunoblotted with the specific antibodies to investigate accumulation of senescent associated proteins p16 INK4A , p21 CIP1 , p53 phosphorylated p53 (S15), Rb and pRb (S807/811). β-Actin served as a loading control. Fold changes in densitometric band intensities for phosphorylated proteins normalized to un-phosphorylated total proteins, acquired by image J were compared. Band intensity of shCntrl was taken as 1. Western blot shown is from replicate from one donor with similar protein expression pattern was observed with 2 different donors. All error bars represents the means ± SEM. p values * = p<0.05, **= p<0.001 and *** = p<.0001.

Article Snippet: Sections were incubated at 4 °C overnight with anti p16 INK4A antibody (1:50) (p16 (clone JC8), Santa Cruz).

Techniques: Infection, Light Microscopy, Expressing, Staining, Control, shRNA, Western Blot

Journal: Aging (Albany NY)

Article Title: Silencing of the small GTPase DIRAS3 induces cellular senescence in human white adipose stromal/progenitor cells

doi: 10.18632/aging.101197

Figure Lengend Snippet: ( A ) (Upper panel) Human shDIRAS3 ASCs (shDIRAS3 hASCs) and shCntrl hASCs were xenotransplanted into posterior sWAT of SCID mice. Injection sites of hASCs were histologically identified and marked by H&E staining. (Lower panel) Senescent DIRAS3 KD hASCs were detected by immunohistochemical staining using anti p16 INK4A antibodies. Region of Interest (ROI) is shown in higher magnification. (B) Number of p16 INK4A positive hASCs per section were counted and plotted (n = 5 per group). (C) (Left panel) Cell lysates from control ASCs and DIRAS3 KD ASCs cultured with and without 20 nM rapamycin were blotted for p16 INK4A , Rb, pRb (S807/811), P70S6K and pP70S6K (T389) using specific antibodies. β-Actin served as loading control. Note, rapamycin was added 2 days after virus infection. (Right panels) Fold changes in densitometric band intensities presented as Arbitrary Units (AU) for phosphorylated proteins normalized to un-phosphorylated total proteins, acquired by image J were compared. Band intensity of shCntrl was taken as 1 (n=2). All error bars represents the means ± SEM. p values * = p<0.05, **= p<0.001 and *** = p<.0001.

Article Snippet: Sections were incubated at 4 °C overnight with anti p16 INK4A antibody (1:50) (p16 (clone JC8), Santa Cruz).

Techniques: Injection, Staining, Immunohistochemical staining, Control, Cell Culture, Virus, Infection

Journal: Aging (Albany NY)

Article Title: Silencing of the small GTPase DIRAS3 induces cellular senescence in human white adipose stromal/progenitor cells

doi: 10.18632/aging.101197

Figure Lengend Snippet: ( A ) (Left panel) Adipogenic differentiation of ASCs was estimated by staining the cells with Oil-Red-O stain at day 9 post induction of differentiation. Non-induced ASC controls are shown. (Right panel) Quantification of Oil-Red-O stained area using image J is shown (n=3). (B) Expression of PPAR γ 2, FABP4 and Perilipin mRNA was estimated at day 3 and day 9 post induction of adipogenesis. Expression at day 0 before induction was taken as 1 and fold increase was calculated. Values are normalized to β Actin. (C) Perilipin and p16 INK4A protein levels were analysed in DIRAS3 KD and control ASCs at day 9 post induction of adipogenesis by western blotting. β-Actin protein served as input control. (D and E) Densitometric evaluation of western blots bands from figure C. Fold changes in densitometric band intensities of perilipin (D) and p16 INK4A (E) normalized to β-Actin protein, acquired by image J were plotted. (F) p16 INK4A mRNA expression was analysed in DIRAS3 KD (red) and control ASCs (black) at day 3 and 9 post adipogenesis induction by q-RT_PCR analysis. All error bars represents the means ± SEM. p values * = p<0.05, **= p<0.001 and *** = p<.0001.

Article Snippet: Sections were incubated at 4 °C overnight with anti p16 INK4A antibody (1:50) (p16 (clone JC8), Santa Cruz).

Techniques: Staining, Expressing, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Aging (Albany NY)

Article Title: Silencing of the small GTPase DIRAS3 induces cellular senescence in human white adipose stromal/progenitor cells

doi: 10.18632/aging.101197

Figure Lengend Snippet: The primer sequences used the present study are indicated

Article Snippet: Sections were incubated at 4 °C overnight with anti p16 INK4A antibody (1:50) (p16 (clone JC8), Santa Cruz).

Techniques: Sequencing

Journal: Journal of cell science

Article Title: Rat aorta-derived mural precursor cells express the Tie2 receptor and respond directly to stimulation by angiopoietins.

doi: 10.1242/jcs.00629

Figure Lengend Snippet: Fig. 5. Tie2 expression in MPCs. (Top) Western-blot analysis (WB) of MPC protein extract immunoreacted for Tie2 demonstrated a 140 kDa band that migrated with the Tie2 band of the endothelial cell control (EC). RT-PCR confirmed expression of Tie2 in MPCs at the mRNA level. Endothelial cells (EC) were used as positive control. No PCR bands were detected in the absence of reverse transcriptase (data not shown). Specificity of the PCR products was confirmed by sequence analysis. (Bottom) Analysis of MPC protein extracts immunoprecipitated (IP) with an anti-Tie2 antibody and reacted with an anti-phosphotyrosine antibody (α-pY) showed phosphorylation of the MPC Tie2 receptor upon stimulation with both Ang-1 and Ang-2. Stripping and reprobing of the membrane with an anti-Tie2 antibody (α-Tie2) showed that equal amounts of Tie2 were present in each lane.

Article Snippet: Tie2 protein expression in isolated cells was demonstrated in separate experiments with two rabbit polyclonal anti- Journal of Cell Science 116 (17) 3637Angiopoietins and Tie2+ mural precursor cells Tie2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; Alpha Diagnostics International, San Antonio, TX).

Techniques: Expressing, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Positive Control, Reverse Transcription, Sequencing, Immunoprecipitation, Phospho-proteomics, Stripping Membranes, Membrane

Journal: Journal of cell science

Article Title: Rat aorta-derived mural precursor cells express the Tie2 receptor and respond directly to stimulation by angiopoietins.

doi: 10.1242/jcs.00629

Figure Lengend Snippet: Fig. 6. Photomicrographs of planar cultures of isolated MPCs (A-E) and endothelial cells (F), and of histologic sections of native rat aorta (G,H) immunostained for Tie2 by immunoperoxidase (A,B,F-H) or immunofluorescence (C-E). Planar cultures: (A) shows a group of confluent MPCs whereas (B) shows a single MPC at higher magnification. Immunoperoxidase staining of cultured cells demonstrated Tie2 in both MPCs (A,B) and control endothelial cells (F). The positive staining reaction for Tie2 was predominantly localized at the cell periphery (arrows). Confocal images of MPCs double stained for Tie2 (C, green fluorescence) and α-SMA (D, red fluorescence) showed coexpression of Tie2 and α-SMA in the same cells (E, green and red fluorescence overlay). Cultured cells reacted with nonimmune IgG were negative (data not shown). Scale bars, 50 µM (A,C-E), 30 µM (B,F). Histological sections of native rat aorta: the intimal and subintimal layers of the aorta contain Tie2+ nonendothelial mesenchymal cells (G,H, arrows). Arrowheads highlight the endothelial lining of the aortic intima, which serves as a positive internal control. Scale bar, 100 µm.

Article Snippet: Tie2 protein expression in isolated cells was demonstrated in separate experiments with two rabbit polyclonal anti- Journal of Cell Science 116 (17) 3637Angiopoietins and Tie2+ mural precursor cells Tie2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; Alpha Diagnostics International, San Antonio, TX).

Techniques: Isolation, Immunoperoxidase Staining, Cell Culture, Control, Staining

Journal: Cancer Communications

Article Title: Targeting autophagy overcomes cancer‐intrinsic resistance to CAR‐T immunotherapy in B‐cell malignancies

doi: 10.1002/cac2.12525

Figure Lengend Snippet: Autophagy limits CAR‐T cell‐mediated cytotoxicity by suppressing TNF‐α induced apoptosis. (A) Volcano plot showing varied genes in Nalm6 cells with the addition of vehicle (as control) and autophinib (log 2 FC > 1 and P < 0.05). (B) Volcano plot showing varied genes between sgControl and indicated RB1CC1 KO Nalm6 cells (log 2 FC > 1 and P < 0.05). (C) KEGG pathway enrichment analysis of varied genes identified in RNA sequencing for Nalm6 cells treated with vehicle (as control) and autophinib. (D) Western blotting showing the expression levels of Caspase‐8, Cleaved caspase‐8, Caspase‐9, Cleaved caspase‐9 and p62 proteins after the addition of vehicle (as control), autophinib and SAR405 in Nalm6 and Raji cells when co‐cultured with or without CD19 CAR‐T cells. GAPDH was used as a loading control. (E) Western blotting showing the expression levels of Caspase‐8, Cleaved caspase‐8, Caspase‐9, Cleaved caspase‐9 and p62 proteins in sgControl and indicated gene‐KO (sgBECN1, sgRB1CC1) Nalm6 and Raji cells when co‐cultured with or without CD19 CAR‐T cells. GAPDH was used as a loading control. (F) Expression of TNFRSF1A mRNA by RT‐qPCR and TNFR1 protein by western blotting in Nalm6 and Raji cells after addition of vehicle (as control), autophinib and SAR405 ( n = 3). Values are shown as the mean ± SD. Statistical differences among three groups in each cell line are calculated with one‐way ANOVA with tests. (G‐H) Cytotoxic analysis of sgControl and indicated gene‐KO (sgTNFRSF1A) Nalm6 and Raji cells co‐cultured with CD19 CAR‐T cells (E:T ratio = 1:4) when treated with vehicle (as control), autophinib and SAR405 and then with or without TNF‐block ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA with tests. (I) Cytotoxic analysis of sgControl and indicated gene‐KO (sgBECN1, sgRB1CC1, sgTNFRSF1A) Nalm6 and Raji cells co‐cultured with CD19 CAR‐T cells (E: T ratio = 1: 4) when treated with or without TNF block ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA with tests. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0,001; ns: not significant. Abbreviations: ANOVA analysis of variance; CAR‐T chimeric antigen receptor T; E:T effector:target; FC fold change; FC fold change; KO knockout; KEGG Kyoto Encyclopedia of Genes and Genomes; RT‐qPCR real‐time quantitative polymerase chain reaction; ns: not significant; SD standard deviation; sg single guide; TNF tumor necrosis factor; TNFR1 tumor necrosis factor receptor 1.

Article Snippet: The following antibodies were used for Western blotting: ATG3 (Abcam, ab108282, 1:500, Oxford, England), RB1CC1 (Proteintech, 17250‐1‐AP, 1:2,000, Rosemont, USA), BECN1 (Proteintech, 11306‐1‐AP, 1:1,000, Rosemont, USA), p62 (Cell Signaling, #5114, 1:1,000, Boston, IL, USA), LC3B (Cell Signaling, #43566, 1:1,000, Boston, IL, USA), TNFR1 (Proteintech, 21574‐1‐AP, 1:1,000, Rosemont, USA), pSTAT1 (Cell Signaling, #9167, 1:1,000, Boston, IL, USA), STAT1 (Cell Signaling, #14994, 1:1,000, Boston, IL, USA), IRF1 (Proteintech, 11335‐1‐AP, 1:1,000, Rosemont, USA), IRF4 (Proteintech, 11247‐1‐AP, 1:1,000, Rosemont, USA), IRF7 (Abcam, ab238137, 1:1,000, Oxford, England), Caspase‐8 (Proteintech, 66093‐1‐Ig, 1:1,000, Rosemont, USA), Caspase‐9 (Proteintech, 66169‐1‐Ig, 1:1,000, Rosemont, USA), cleaved Caspase‐8 (Cell Signaling, # 9748, 1:1,000, Boston, IL, USA), cleaved Caspase‐9 (Abcam, ab2324, 1:1,000, Oxford, England) and GAPDH (Proteintech, 60004‐1‐Ig, 1:8,000, Rosemont, USA).

Techniques: Control, RNA Sequencing, Western Blot, Expressing, Cell Culture, Quantitative RT-PCR, Blocking Assay, Knock-Out, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting autophagy overcomes cancer‐intrinsic resistance to CAR‐T immunotherapy in B‐cell malignancies

doi: 10.1002/cac2.12525

Figure Lengend Snippet: STAT1/IRF1 axis mediates the upregulation of CXCL10 and CXCL11 induced by autophagy targeting. (A‐B) Western blotting showing the expression levels of STAT1, pSTAT1, and IRF1 proteins in Nalm6 and Raji cells after the addition of autophagy inhibitors or the knockout of RB1CC1 ( n = 3). GAPDH was used as a loading control. The histograms showing the expression of STAT1 and IRF1 mRNA by RT‐qPCR quantification. Values are shown as the mean ± SD. Statistical differences are calculated with one‐way ANOVA tests. (C‐D) Western blotting showing the expression levels of STAT1 and IRF1 proteins in Nalm6 and Raji cells after the addition of autophagy inhibitors and the silencing of STAT1 ( n = 3). GAPDH was used as a loading control. The histograms showing the expression of STAT1 and IRF1 mRNA by RT‐qPCR quantification. Values are shown as the mean ± SD. Statistical differences are calculated with one‐way ANOVA tests. (E‐F) Expression of CXCL10 and CXCL11 mRNA by RT‐qPCR and ELISA quantification of CXCL10 and CXCL11 protein levels in the supernatants of shControl, shSTAT1 and shIRF1 Nalm6 and Raji cells ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA tests. (G‐H) ChIP‐qPCR showing the binding of STAT1 and IRF1 to the promoter region of CXCL10 and CXCL11 ( n = 3). Values are shown as the mean ± SD. Statistical differences for each cell line are calculated with unpaired Student's t tests. (I) Graphic abstract: in the proposed model, inhibition of cancer cell‐autonomous autophagy leads to accumulation of cytosolic DNA, which thereby not only suppresses cancer cell survival by inducing TNFR1‐TNF‐α mediated apoptosis but also promotes the CAR‐T cell recruitment in tumor microenvironment via STAT1/IRF1‐dependent activation of chemokine signaling. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0,001; ns: not significant. Abbreviations: ANOVA analysis of variance; CAR‐T, chimeric antigen receptor T; ChIP, chromatin immunoprecipitation; CXCL CXC, chemokine ligand; ELISA, enzyme‐linked immunosorbent assay; FC, fold change; IRF, interferon regulatory factor; ns: not significant; RT‐qPCR, real‐time quantitative polymerase chain reaction; SD, standard deviation; sh short hairpin; STAT, signal transducers and activators of transcription.

Article Snippet: The following antibodies were used for Western blotting: ATG3 (Abcam, ab108282, 1:500, Oxford, England), RB1CC1 (Proteintech, 17250‐1‐AP, 1:2,000, Rosemont, USA), BECN1 (Proteintech, 11306‐1‐AP, 1:1,000, Rosemont, USA), p62 (Cell Signaling, #5114, 1:1,000, Boston, IL, USA), LC3B (Cell Signaling, #43566, 1:1,000, Boston, IL, USA), TNFR1 (Proteintech, 21574‐1‐AP, 1:1,000, Rosemont, USA), pSTAT1 (Cell Signaling, #9167, 1:1,000, Boston, IL, USA), STAT1 (Cell Signaling, #14994, 1:1,000, Boston, IL, USA), IRF1 (Proteintech, 11335‐1‐AP, 1:1,000, Rosemont, USA), IRF4 (Proteintech, 11247‐1‐AP, 1:1,000, Rosemont, USA), IRF7 (Abcam, ab238137, 1:1,000, Oxford, England), Caspase‐8 (Proteintech, 66093‐1‐Ig, 1:1,000, Rosemont, USA), Caspase‐9 (Proteintech, 66169‐1‐Ig, 1:1,000, Rosemont, USA), cleaved Caspase‐8 (Cell Signaling, # 9748, 1:1,000, Boston, IL, USA), cleaved Caspase‐9 (Abcam, ab2324, 1:1,000, Oxford, England) and GAPDH (Proteintech, 60004‐1‐Ig, 1:8,000, Rosemont, USA).

Techniques: Western Blot, Expressing, Knock-Out, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, Binding Assay, Inhibition, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Hepatocyte-specific Wtap deficiency promotes hepatocellular carcinoma by activating GRB2–ERK depending on downregulation of proteasome-related genes

doi: 10.1016/j.jbc.2023.105301

Figure Lengend Snippet: Wtap deletion in hepatocytes promotes hepatocellular carcinoma (HCC) progression in young mice. At 2 weeks old, male Wtap -HKO mice and Wtap flox/flox mice received a single intraperitoneal dose of DEN (50 mg/kg). Mice were fed either a normal choe (NC) diet or a high-fat diet (HFD) for 11 weeks. A , representative pictures of mouse livers from the indicated genotype are shown. B , tumor number was counted (n = 10–12 per group; NC: p = 0.0026; HFD: p < 0.0001). C , maximal tumor size was measured (n = 10–12 per group; NC: p < 0.0001; HFD: p < 0.0001). D , representative Ki67 staining photographs were shown. E , the number of Ki67-positive cells was counted (n = 6 per group; NC: p = 0.0002; HFD: p < 0.0001). F and G , triglyceride (TAG) levels in the livers of Wtap -HKO mice and Wtap flox/flox mice fed an NC diet for 35 weeks or an HFD for 11 weeks were assessed (n = 10 per group; F : p < 0.0001; G : p = 0.0012). H , serum-free fatty acid levels in Wtap -HKO mice and Wtap flox/flox mice fed with an NC diet for 11 weeks (n = 6–8 per group; p = 0.0022). I and J , immunoblotting of IGFBP1, CD36, CCL2, WTAP, and Tubulin in the livers of Wtap -HKO and Wtap flox/flox mice fed NC or HFD for 11 weeks following DEN treatment (n = 7 per group; n = 4 for representative images; NC: IGFBP1, p = 0.0033; CD36, p = 0.0013; and CCL2, p = 0.019; HFD: IGFBP1, p = 0.0115; CD36, p = 0.0264; and CCL2, p = 0.0011). K , heatmap of interleukin 6 (IL6) cytokine family ( Clcf1 , Cntf , Ctf1 , Il6 , IL11 , Il27 , Lif , and Osm ) and Tnfa in Wtap -HKO and Wtap flox/flox livers (n = 3 for each group, RNA-Seq data GSE168850). L , immunoblotting analysis of p-STAT3, STAT3, p-p65, p65, p-IκBα, IκBα, WTAP, and Tubulin in the livers of NC-fed Wtap -HKO and Wtap flox/flox mice pretreated with or without DEN (n = 7 for each group; n = 3 for representative images). ∗ p < 0.05; ∗∗ p < 0.01. Data represent the mean ± SD. CCL2, chemokine (C–C motif) ligand 2; DEN, diethylnitrosamine; IGFBP1, insulin-like growth factor–binding protein 1; Wtap -HKO, hepatocyte-specific Wtap knockout.

Article Snippet: The following antibodies were displayed: FLAG (catalog no.: F1804, 1:5000 dilution; Sigma); WTAP (catalog no.: 10200-1-AP, 1:2000 dilution; Proteintech); PSMB4 (catalog no.: 11029-1-AP, 1:5000 dilution; Proteintech); PSMB6 (catalog no.: 11684-2-AP, 1:5000 dilution; Proteintech); p-STAT3 (catalog no.: 9145, 1:3000 dilution; Cell Signaling Technology); STAT3 (catalog no.: 10253-2-AP, 1:3000 dilution; Proteintech); p-IκBα (Ser32) (catalog no.: 2859, 1:3000 dilution; Cell Signaling Technology); IκBα (catalog no.: 10268-1-AP, 1:2000 dilution; Proteintech); Tubulin (catalog no.: sc-5286, 1:5000 dilution; Santa Cruz); CD36 (catalog no.: 18836-1-AP, 1:2500 dilution; Proteintech); CCL2 (catalog no.: 66272-1-Ig, 1:2000 dilution; Proteintech); p-p65 (catalog no.: 3033, 1:2500 dilution; Cell Signaling Technology); p65 (catalog no.: 8242, 1:2500 dilution; Cell Signaling Technology); p-ERK1/2 (catalog no.: 4377, 1:5000 dilution; Cell Signaling Technology); ERK1/2 (catalog no.: 4695, 1:5000 dilution; Cell Signaling Technology); GRB2 (catalog no.: sc-8034, 1:1000 dilution; Santa Cruz); p-Met (catalog no.: 3077, 1:4000 dilution; Cell Signaling Technology); Met (catalog no.: 3127, 1:4000 dilution; Cell Signaling Technology); p-EGFR (catalog no.: 3777, 1:4000 dilution; Cell Signaling Technology); EGFR (catalog no.: 4267, 1:5000 dilution; Cell Signaling Technology); p-MEK1/2 (catalog no.: 9154, 1:4000 dilution; Cell Signaling Technology); MEK1/2 (catalog no.: 8727, 1:4000 dilution; Cell Signaling Technology); and IGFBP1 (catalog no.: A11672, 1:3000 dilution; ABclonal).

Techniques: Staining, Western Blot, RNA Sequencing, Binding Assay, Knock-Out

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 was highly expressed in macrophages within the metastatic lesions of gastric cancer. ( A ) UMAP representation colored according to different cell types. ( B ) Density plot of SPI1 expression distribution in scRNA sequencing data. ( C ) UMAP indicated SPI1 staining of macrophages in primary and metastasis gastric cancer (GC). ( D ) Differential analysis of SPI1 expression in the primary and metastasis tumor of GC in scRNA sequencing data. ( E ) Infiltration of SPI1 + CD68 + TAMs in normal tissue, primary tumor, and metastasis sites of GC. ( F ) Differential analysis of SPI1 + CD68 + TAMs infiltration between primary and metastasis tumor according to double immunohistochemical staining. ( G ) SPI1 + CD68 + TAMs were screened by flow cytometry in primary and metastasis tumor of patients with GC. ( H ) Quantitative analysis of SPI1 + CD68 + TAMs infiltration based on flow cytometry. scRNA, single-cell RNA; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Expressing, Sequencing, Staining, Immunohistochemical staining, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 + CD68 + TAMs was an independent prognostic factor in patients with metastatic gastric cancer. ( A ) Overall survival (OS) and disease-free survival (DFS) of patients with gastric cancer (GC) in different SPI1 + CD68 + TAMs groups in the training cohort. ( B ) OS and DFS of patients with GC in different SPI1 + CD68 + TAMs groups in the external validation cohort. ( C ) OS and DFS of patients with GC in different SPI1 − CD68 + TAMs groups in the training cohort. ( D ) OS and DFS of patients with GC in different SPI1 − CD68 + TAMs groups in the external validation cohort. ( E ) Univariate cox regression analysis of patients with GC in the training cohort. ( F ) SPI1 + CD68 + TAMs was an independent prognostic factor of patients with GC in the training cohort. ( G ) Univariate cox regression analysis of patients with GC in the external validation cohort. ( H ) SPI1 + CD68 + TAMs was an independent prognostic factor of patients with GC in the external validation cohort. SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Biomarker Discovery

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 was associated with M2 polarization of macrophages. ( A ) Screening M1/M2 macrophages used flow cytometry with CD45, CD68, CD80, CD206 and SPI1 in patients with gastric cancer (GC). ( B ) Quantitative analysis of SPI1 + CD68 + TAMs infiltration in M1 and M2 type based on flow cytometry of patients with GC. ( C ) The markers of induced M1 and M2-type macrophages were detected by qRT-PCR. ( D ) Detecting the macrophage-related markers with qRT-PCR after SPI1 knockdown. ( E ) qRT-PCR was used to detect the macrophage-associated markers after SPI1 overexpression. ( F ) Western blot was used to detect the macrophage-related markers after SPI1 knockdown. ( G ) Macrophage-related markers were detected with western blot after SPI1 overexpression. ( H ) Flow cytometry was used to detect the macrophage-related markers after SPI1 knockdown. ( I ) Quantitative analysis of flow cytometry in SPI1 knockdown macrophages. ( J ) Flow cytometry was used to detect the macrophage-related markers after SPI1 overexpression. ( K ) Quantitative analysis of flow cytometry in SPI1 overexpression macrophages. IL-10, interleukin-10; mRNA, messenger RNA; qRT-PCR, quantitative Reverse Transcription Polymerase Chain Reaction; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Flow Cytometry, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 + CD68 + TAMs promoted tumor angiogenesis through VEGF pathway. ( A ) GSVA analysis between SPI1-high and SPI1-low TAMs groups in scRNA sequencing data. ( B ) Incoming and outgoing interaction strength in different types of cells. ( C ) Cell–cell communication between different cell types in the VEGF signaling pathway. ( D ) Response to bevacizumab in patients with different TAMs infiltrates in the external validation cohort. ( E ) Immunofluorescence revealed the infiltration of SPI1 + CD68 + TAMs around the tumor blood vessels (CD31 marked). ( F ) Difference in the number of SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs around tumor vessels. ( G ) Difference in distance between blood vessels with SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs. DAPI, 4',6-Diamidino-2-Phenylindole; GSVA, gene set variation analysis; NK, Natural Killer cells; scRNA, single-cell RNA; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages; VEGFA, vascular endothelial growth factor A.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Sequencing, Biomarker Discovery, Immunofluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: The role of SPI1 in GC cell growth and metastasis in vivo. ( A ) The representative images of the xenograft tumor. ( B ) Quantitative analysis of tumor growth curve. ( C ) Quantitative analysis of tumor weight in different SPI1 expression group. ( D ) Bioluminescence images of tumor-bearing mice individually treated with shNC, shSPI1, Vector, SPI1 transfected macrophages at day 5, 10, 15, 20, 25, 30, 40, and 50. ( E ) Representative photographs of peritoneum and mesentery metastasis lesions in different SPI1 expression groups. ( F ) The fluorescence intensity of tumors in various groups of mice. ( G ) Quantitative analysis of peritoneum nodules. ( H ) Survival curves of mice in different groups. GC, gastric cancer; SPI1, Spi-1 proto-oncogene.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: In Vivo, Expressing, Plasmid Preparation, Transfection, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 closely interacted with endothelial cells and regulated VEGFA transcription. ( A ) Wound healing assay was used to detect the migration ability of HUVEC cells cultured with conditioned medium of SPI1 knockdown macrophages. ( B ) Compared with the control group, the overexpression of SPI1 enhanced the migration ability of HUVEC cells. ( C ) Tube formation assay was used to detect the angiogenic ability of HUVEC cells after cultured with conditioned medium of SPI1 knockdown macrophages. ( D ) On overexpressing SPI1, the angiogenesis ability of HUVEC cells was augmented. ( E ) The mRNA level of VEGFA changed after knockdown or overexpression of SPI1. ( F ) The protein level of VEGFA changed after knockdown or overexpression of SPI1. ( G ) Motif sequence logo plot of SPI1 according to JASPAR database. ( H ) Putative SPI1 and VEGFA promoter binding sites. ( I ) ChIP analysis of the direct interaction between SPI1 and the promoter of VEGFA. ( J ) The binding site of SPI1 to the VEGFA promoter was assessed using ChIP-qPCR. ChIP, chromatin immunoprecipitation; HUVEC, Human Umbilical Vein Endothelial Cells; mRNA, messenger RNA; qPCR, quantitative PCR; SPI1, Spi-1 proto-oncogene; VEGFA, vascular endothelial growth factor A.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Wound Healing Assay, Migration, Cell Culture, Knockdown, Control, Over Expression, Tube Formation Assay, Sequencing, Binding Assay, ChIP-qPCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: In vivo response to anti-PD-1 immunotherapy and antiangiogenesis treatment. ( A ) Response to immunotherapy in patients with different CPS scores and SPI1 + CD68 + TAMs infiltration. ( B ) Difference analysis of immunotherapy response in patients with different SPI1 + CD68 + TAMs infiltration. ( C ) Correlation analysis of SPI1 and PD-L1. ( D–G ) ROC curve of CPS score, CD68 + cells, SPI1 + CD68 + TAMs, and CPS score plus SPI1 + CD68 + TAMs. ( H ) Multiple immunofluorescences staining of PD-1 + CD8 + T cells and SPI1 + CD68 + TAMs. ( I ) Spatial distribution of SPI1 + CD68 + TAMs, SPI1 − CD68 + TAMs and PD-1 + CD8 + T cells analyzed by HALO. ( J ) Average distance from PD-1 + CD8 + T cells to SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs (p<0.05). ( K ) The discrepancy in the quantity of SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs surrounding PD-1 + CD8 + T cells (p<0.05). ( L ) Bioluminescence images of NOD/SCID mice with different treatment in week 1, 3, and 6. ( M ) The fluorescence intensity of intraperitoneal tumors in various groups of mice. CPS, Combined Positive Score; DAPI, 4',6-Diamidino-2-Phenylindole; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand 1; ROC, receiver operating characteristic; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: In Vivo, Staining, Fluorescence

Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-CHAMP5 antibody Clone F-7 , Santa Cruz Biotechnology , Cat# sc-374338; RRID:AB_10989738.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Protease Inhibitor, SYBR Green Assay, Purification, Gel Extraction, Reverse Transcription, Quantitative RT-PCR, Plasmid Preparation, Isolation, Cell Culture, Immunoprecipitation, Gene Expression, CRISPR, Software, Sequencing, Modification

Journal: Biochimica et biophysica acta

Article Title: Post-transcriptional modulation of interleukin 8 by CNOT6L regulates skeletal muscle differentiation.

doi: 10.1016/j.bbamcr.2015.11.018

Figure Lengend Snippet: Fig. 2. IL-8 mRNA is a target of CNOT6L. A. IL-8 mRNA expression in proliferating (P) and differentiating LHCN human myoblasts. IL-8 mRNA amount was normalized to cyclophilinA and expressed as a relative amount to P condition. Results are presented as the mean ± SD of triplicates. B. Quantification of IL-8 mRNA bound by CNOT6L in C2C12 cells. Following 2 days of differentiation, relative IL8 mRNA levels were quantified by RT-qPCR in FH-CNOT6L transfected cells and in control cells after FLAG-HA immunoprecipitation. IL-8 amount was normalized to total RNA and expressed as relative amount to pRev condition. Results are expressed as the mean ± SD of 4 experiments. Lower panel: representative WB with HA antibody for eluted exogenous CNOT6L after FLAG-HA immunoprecipitation. C. Stability of IL-8 mRNA. LHCN cells were transfected with CNOT6L-targeting siRNA (no. 1 or 2) or a control siRNA at day 0. Cells were placed in differentiation conditions for 48 h and treated with DRB (5, 6-Dichlorobenzimidazole riboside) for the indicated amount of time. IL-8 mRNA was quantified as described in A and reported as a relative expression to T0. Results are expressed as the mean ± SD of 3 independent experiments performed in duplicates. D. Poly(A) tail length assay of IL-8 mRNA. LHCN cells were transfected with CNOT6L-targeting siRNA or a control siRNA, placed in differentiation conditions and total mRNAs were extracted. IL-8 coding sequence (CS) was amplified by RT-PCR and IL-8 CS amplicon amount (normalized to that of CyclophilinA) was used to adjust the quantity of IL-8 mRNA used in PAT assay. Poly(A) tail length of IL-8 mRNA was evaluated by RT-PCR using 2 different primers (1 or 2) and size of minimum PCR product is indicated. Right panel: representative pattern on 2% agarose gel. Statistical significance was evaluated by bilateral unpaired two-sample Student's t-test. ⁎⁎⁎p b 0001, ⁎⁎p b 0.01, ⁎p b 0.05.

Article Snippet: Antibodies against beta-actin (Sigma, A5441), human CNOT6L (ab103659, Abcam), mouse CNOT6L (Santa Cruz Biotechnology, sc246,304), MyoD (Santa Cruz Biotechnology, sc-760) MCK (creatine kinase-M, Santa Cruz Biotechnology, sc-15,161), HA (Sigma, H3663), and HRP-conjugated secondary antibodies (Sigma, A2304 and A0545) were used in Western blots.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Immunoprecipitation, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

Journal: Biochimica et biophysica acta

Article Title: Post-transcriptional modulation of interleukin 8 by CNOT6L regulates skeletal muscle differentiation.

doi: 10.1016/j.bbamcr.2015.11.018

Figure Lengend Snippet: Fig. 3. IL-8, a novel CNOT6L target, controls muscle cell differentiation. A. IL-8 protein expression in supernatant of proliferating (P) and differentiating LHCN human myoblasts. IL-8 protein amount (μg/mL) was quantitated by ELISA in the supernatants of LHCN cell cultures over the course of differentiation. Results are expressed as the relative amount of IL-8 compared to P condition (mean ± SD of three independent replicates). B–C. Functional role of IL-8 on LHCN differentiation by gain- or loss-of-function assays. Differentiation of LHCN was induced with or without recombinant IL-8 (3 μg/mL) for gain-of-function assay (B) or with IL-8 neutralizing or isotypic antibodies at the indicated concentrations for loss-of-function assay (C). Differen- tiation of LHCN was monitored quantifying the number of myotubes at 5 (B) or 7 (C) days of differentiation as described in Fig. 1D. Left panels: typical acquisition fields. Right panels: Data are expressed as the mean ± SD of 3 independent experiments performed in triplicates. D. IL-8 protein level in CNOT6L KD LHCN cell culture supernatants. LHCN cells were transfected with a CNOT6L-targeting siRNA (no. 1 or 2) or a control siRNA. After 3 days of differentiation, IL-8 protein level was quantified by ELISA. Results are expressed as the mean ± SD of two independent experiments performed in triplicates. E. LHCN cells were transfected with CNOT6L-targeting siRNA or a control siRNA. Differentiation was assayed as described in B–C, with neutralizing IL-8 or isotypic antibody. Data are expressed as the mean ± SD of 3 independent experiments performed in triplicates. Statistical significance was evaluated by bilateral un- paired two-sample Student's t-test ⁎⁎⁎p b 0001, ⁎⁎p b 0.01, ⁎p b 0.05.

Article Snippet: Antibodies against beta-actin (Sigma, A5441), human CNOT6L (ab103659, Abcam), mouse CNOT6L (Santa Cruz Biotechnology, sc246,304), MyoD (Santa Cruz Biotechnology, sc-760) MCK (creatine kinase-M, Santa Cruz Biotechnology, sc-15,161), HA (Sigma, H3663), and HRP-conjugated secondary antibodies (Sigma, A2304 and A0545) were used in Western blots.

Techniques: Cell Differentiation, Expressing, Enzyme-linked Immunosorbent Assay, Functional Assay, Recombinant, Cell Culture, Transfection, Control