Journal: Cell Death & Disease

Article Title: OTUD7B stabilizes estrogen receptor α and promotes breast cancer cell proliferation

doi: 10.1038/s41419-021-03785-7

Figure Lengend Snippet: A An immunofluorescence assay demonstrated that OTUD7B and ERα at least partially colocalized in MCF7 and T47D cells. B Co-IP assay revealed an association between endogenous OTUD7B and ERα in MCF-7 cells. MCF-7 cells were harvested with RIPA lysis buffer. Co-IP was performed using antibody as indicated. C Purified His-OTUD7B was incubated with GST- ERα or GST protein. The interacted OTUD7B was detected via western blot. D ER alpha domain structure and deletion mutants used in the study. E , F OTUD7B interacted with ERα through its AF1 domain. HEK293 cells were transfected with 2 μg Myc-OTUD7B together with HA- ERα full length or mutants. After 24 h, cells were harvested with NP-40 lysis buffer. Co-IP was performed using Myc antibody. The possible interacted ERα domains were detected by HA antibody.

Article Snippet: HEK293 cells were transfected with HA-Ub, Flag-ERα, Myc-OTUD7B, or Myc-OTUD7B C194S plasmid as indicated for 48 h. After 6-h incubation with 10 μM MG132 (MCE), cells were harvested and immunoprecipitated to isolate HA-ubiquitinated ERα.

Techniques: Immunofluorescence, Co-Immunoprecipitation Assay, Lysis, Purification, Incubation, Western Blot, Transfection

Journal: Cell Death & Disease

Article Title: OTUD7B stabilizes estrogen receptor α and promotes breast cancer cell proliferation

doi: 10.1038/s41419-021-03785-7

Figure Lengend Snippet: A In the presence of the proteasome inhibitor MG132, depletion of OTUD7B did not further decrease the ERα protein level. Breast cancer cells were transfected with siOTUD7B or siControl. After 48 h, cells were treated with 10 µM MG132/vehicle for 6 h; cell lysates were prepared for western blot analysis. B MCF-7 cells were transfected with OTUD7B (wild type or C194S) together with OTUD7B siRNA. The ERα levels were measured. C OTUD7B depletion decreased ERα half-life in breast cancer cells. Breast cancer cells were transfected with siOTUD7B or siControl. After 48 h, cells were treated with 100 µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for western blot analysis. D OTUD7B C194S did not increase ERα half-life in HEK293 cells. HEK293 cells were transfected with HA-ERα plasmid and Myc-tag, Myc-OTUD7B, or Myc-OTUD7B C194S plasmids. After 24 h, cells were treated with 100-µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for Western blot analysis.

Article Snippet: HEK293 cells were transfected with HA-Ub, Flag-ERα, Myc-OTUD7B, or Myc-OTUD7B C194S plasmid as indicated for 48 h. After 6-h incubation with 10 μM MG132 (MCE), cells were harvested and immunoprecipitated to isolate HA-ubiquitinated ERα.

Techniques: Transfection, Western Blot, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: OTUD7B stabilizes estrogen receptor α and promotes breast cancer cell proliferation

doi: 10.1038/s41419-021-03785-7

Figure Lengend Snippet: A MCF-7 cells transfected with the indicated siRNA were treated with MG132 for 6 h before collection. ERα was immunoprecipitated with anti-ERα and immunoblotted with anti-HA. B Immunoblotting to detect the ubiquitination of ERα in HEK293 cells cotransfected with Flag-ERα, HA-Ubiquitin, and Myc-OTUD7B (wild type or C194S). C Immunoblotting to detect the ubiquitination of ERα in HEK293 cells cotransfected with Flag-ERα, HA-Ubiquitin, and Myc-OTUD7B (wild type or C194S). D OTUD7B removed the ubiquitin chain of ERα in a dose-dependent manner. E OTUD7B removed the ubiquitin chain of ERα in a time- and dose-dependent manner. F ERα ubiquitylation was analyzed in cells transfected with E3 TRIM8 together with OTUD7B or not. G HA-WT, K6, K11, K27, K29, K33, and K48 or K63 Ub was cotransfected with Flag-ERα and Myc-OTUD7B into HEK293 cells. After treatment with 10 μM MG132 for 6 h, cell lysates were subjected to ubiquitination assay and the ubiquitination level of ERα was detected by HA antibody. * P value < 0.05, ** P value < 0.01, *** P value < 0.001.

Article Snippet: HEK293 cells were transfected with HA-Ub, Flag-ERα, Myc-OTUD7B, or Myc-OTUD7B C194S plasmid as indicated for 48 h. After 6-h incubation with 10 μM MG132 (MCE), cells were harvested and immunoprecipitated to isolate HA-ubiquitinated ERα.

Techniques: Transfection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: IL-17A is increased in diabetic wounds and impairs keratinocyte function via histone demethylase JMJD3

doi: 10.1038/s41467-025-67456-3

Figure Lengend Snippet: a ChIP analysis of H3K27me3 or IgG at indicated promoters in keratinocytes with (white) or without (blue) IL-17A stimulation. Itga3: n = 5 (unstimulated), n = 4 (IL-17A-stimulated) technical replicates, p = 0.0034, Timp1 : n = 3 technical replicates, p = 0.0046, Ccl20 : n = 3 technical replicates, p = 0.0059, Cxcl1 : n = 3 technical replicates, p = 0.0020, Cxcl3 : n = 3 technical replicates, p = 0.0258, Cxcl5 : n = 3 technical replicates, p = 0.0174. n = 3 independent experiments. b qPCR analysis of keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (1 µM) (red). Itga3 : n = 4 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0017 (IL-17A vs. IL-17A and inhibitor), Timp1: n = 6 biological replicates, p < 0.0312 (DMSO vs. IL-17A), p = 0.0029 (IL-17A vs. IL-17A and inhibitor), Ccl20: n = 3 biological replicates, p = 0.0008 (DMSO vs. IL-17A), p = 0.0428 (IL-17A vs. IL-17A and inhibitor), Cxcl1 : n = 3 biological replicates, p = 0.0003 (DMSO vs. IL-17A), p = 0.0294 (IL-17A vs. IL-17A and inhibitor), Cxcl3 : n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0003 (IL-17A vs. IL-17A and inhibitor), Cxcl5 : n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0007 (IL-17A vs. IL-17A and inhibitor), n = 3 independent experiments. c Western blot of ITGA-3 expression in keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (red). Representative densitometry plot is shown. n = 3 independent experiments. d Protein quantification of lysates from keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (red). TIMP-1: n = 6 biological replicates, p = 0.0012 (DMSO vs. IL-17A), p = 0.0068 (IL-17A vs. IL-17A and inhibitor), CCL-20: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0199 (IL-17A vs. IL-17A and inhibitor), CXCL-1: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0158 (IL-17A vs. IL-17A and inhibitor), CXCL-3: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0167 (IL-17A vs. IL-17A and inhibitor), CXCL-5: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0005 (IL-17A vs. IL-17A and inhibitor), n = 3 independent experiments. e qPCR analysis of keratinocytes treated with a non-targeting control (siNTC) (white) or si Jmjd3 (gray). Jmjd3 : n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0192, Itga3: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0019, Timp1: n = 3 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0205, Ccl20: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0015, Cxcl1: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0019, Cxcl3: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0011, Cxcl5: n = 6 (siNTC), n = 4 biological replicates (si Jmjd3 ), p = 0.0011, n = 3 independent experiments. f qPCR analysis of Jmjd3 fl/fl K14 cre+ (red) and Jmjd3 fl/fl K14 cre- (yellow) keratinocytes. n = 3 biological replicates, Itga3 : p = 0.0184, Timp1 : p = 0.0044, Ccl20 : p = 0.0160, Cxcl1 : p = 0.0371, Cxcl3 : p = 0.0042. n = 3 independent experiments. g , h Scratch assays of primary murine ( n = 3 biological replicates, p < 0.0001 (48 h)) and N/TERT ( n = 3 biological replicates, p = 0.0340 (12 h)) keratinocytes treated with IL-17A alone (blue) or IL-17A and GSK-J4 (red). n = 3 independent experiments. i , j Scratch assays of primary murine ( n = 3 biological replicates, p = 0.0016 (48 h)) and N/TERT ( n = 6 biological replicates (IL-17A alone), n = 4 biological replicates (IL-17A and GSK-J1), p = 0.0223 (8 h), p < 0.0001 (12 h)) keratinocytes treated with IL-17A alone (blue) or IL-17A and GSK-J1 (red). n = 3 independent experiments. k Scratch assay of Jmjd3 fl/fl K14 cre+ (red) and Jmjd3 fl/fl K14 cre- (blue) keratinocytes. n = 3 biological replicates, p = 0.0198 (48 h). n = 3 independent experiments. Data were analyzed for variances, and 2-tailed Student’s t tests for ( a ), ( e ), ( f ) and 1-way ANOVA tests for ( b ), ( d ), ( g – k ) were performed. Data are presented as the mean ± SEM.

Article Snippet: After stimulation, cell free supernatant was collected and analyzed by the University of Michigan Immune Monitoring Shared Resource Core for CCL-20, CXCL-1, CXCL-5 or specific enzyme immunoassay kits for CXCL-3 (Boster Bio) and TIMP-1 (R&D Systems) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Wound Healing Assay

Journal:

Article Title: Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells

doi: 10.1073/pnas.261562798

Figure Lengend Snippet: Heterogeneous expression of Flk-2 on the HSC population. (A) Flk-2 expression was analyzed on the HSC population; Lin−/lo Thy-1.1lo c-kit+ Sca-1+ of c-kit enriched BM. HSCs that were either Flk-2+ or Flk-2− were sorted. Lethally irradiated recipients were competitively reconstituted with 50–100 (B) or 20 (C) Flk-2+ or Flk-2− HSCs plus host-type radioprotective WBM. (B) Fifty Flk-2+ HSCs are shown in blue, 100 flk-2+ HSCs are in light blue, 50 Flk-2− HSCs are in red, and 100 Flk-2− HSCs are in orange. (C) Twenty Flk-2+ HSCs are shown in blue, and 20 Flk-2− HSCs are in red. Peripheral blood was analyzed for donor Ly5 B, T, and myeloid contribution. Each curve represents an individual animal. Because myeloid cells are present in the blood for a short period, myeloid activity is shown as a good indicator of stem cell activity. The background is 0.1%.

Article Snippet: A20.1 (anti-Ly5.1, FITC-conjugated, Becton Dickenson–PharMingen), and AL1–4A2 (anti-Ly5.2, Texas red conjugate) were used to analyze donor cells after reconstitution.

Techniques: Expressing, Irradiation, Activity Assay

Journal:

Article Title: Hematopoietic potential of stem cells isolated from murine skeletal muscle

doi:

Figure Lengend Snippet: Flow cytometric analysis of peripheral blood from a representative mouse transplanted with muscle cells. Peripheral blood was drawn from the transplant recipient 6 weeks after it received 18 × 103 Ly-5.1+ mononuclear muscle cells and 200 × 103 Ly-5.2+ whole bone marrow cells and then was stained with anti-Ly-5.1-biotin followed by Str-PE and antibodies to specific lineage markers. (A–C) Controls. (B–F) Peripheral blood from transplant recipient. (A) C57BL/6-Ly-5.2 peripheral blood stained with anti-Ly-5.1-biotin followed by Str-PE. (B) C57BL/6-Ly-5.1 peripheral blood stained with anti-Ly-5.1-biotin + Str-PE. (C) C57BL/6-Ly-5.1 peripheral blood stained with isotype controls for lineage markers (a mixture of rat-IgG2a-FITC and rat-IgG2b-FITC). (D) Anti-B220-FITC (B cells) with anti-Ly-5.1-biotin + Str-PE. (E) Anti-Thy-1-FITC (T cells) with anti-Ly-5.1 biotin + Str-PE. (F) Anti-Gr-1-FITC + anti-Mac-1-FITC (granulocytes and macrophages, respectively) with anti-Ly-5.1-biotin + Str-PE. The percentage of cells in each quadrant is indicated in the Upper Right corner. Bracketed numbers (D–F) are the percentages of lineage-positive cells derived from muscle. This calculation was made by dividing the number of cells in the Upper Right quadrant by the total number of lineage-positive cells (the sum of both Right quadrants).

Article Snippet: The nucleated cells were then stained with anti-Ly-5.1-biotin (clone A20), rat-IgG2a-FITC (R35–95), rat-IgG2b-FITC (A95–1), B220-FITC (RA3–6B2), Thy-1-FITC (30-H12), Gr-1-FITC (RB6–8C5), and Mac-1-FITC (M1/70) (all from PharMingen).

Techniques: Staining, Derivative Assay

Journal:

Article Title: Hematopoietic potential of stem cells isolated from murine skeletal muscle

doi:

Figure Lengend Snippet: Multilineage engraftment in all mice transplanted with muscle cells. Peripheral blood was drawn 6 and 12 weeks after transplantation and stained with antibodies against the Ly-5.1 marker, B220 (B cells), Thy-1 (T cells), and Gr-1 and Mac-1 (granulocytes and macrophages, respectively). Stained blood samples were analyzed by flow cytometry, as described in Experimental Procedures. The percentages of positive cells in each lineage are reported. Mouse 1 (M1) was killed after the 6-week analysis for subsequent study (Fig. ​(Fig.33).

Article Snippet: The nucleated cells were then stained with anti-Ly-5.1-biotin (clone A20), rat-IgG2a-FITC (R35–95), rat-IgG2b-FITC (A95–1), B220-FITC (RA3–6B2), Thy-1-FITC (30-H12), Gr-1-FITC (RB6–8C5), and Mac-1-FITC (M1/70) (all from PharMingen).

Techniques: Transplantation Assay, Staining, Marker, Flow Cytometry

Journal:

Article Title: Hematopoietic potential of stem cells isolated from murine skeletal muscle

doi:

Figure Lengend Snippet: Repopulation of lymphoid and myeloid cell compartments in secondary recipients of muscle cells. Bone marrow from mouse 1 (M1; Fig. ​Fig.2)2) was transplanted at 8 × 105 nucleated cells per mouse into each of five lethally irradiated C57BL/6-Ly-5.2 recipients. After 5 weeks, peripheral blood was drawn and stained with antibodies against Ly-5.1, B220 (B cells), Thy-1 (T cells), and Gr-1/Mac-1 (granulocytes and macrophages) and then analyzed by flow cytometry. The findings are displayed as the percentages of cells positive for Ly-5.1 and specific lineage markers.

Article Snippet: The nucleated cells were then stained with anti-Ly-5.1-biotin (clone A20), rat-IgG2a-FITC (R35–95), rat-IgG2b-FITC (A95–1), B220-FITC (RA3–6B2), Thy-1-FITC (30-H12), Gr-1-FITC (RB6–8C5), and Mac-1-FITC (M1/70) (all from PharMingen).

Techniques: Irradiation, Staining, Flow Cytometry

Journal:

Article Title: Hematopoietic potential of stem cells isolated from murine skeletal muscle

doi:

Figure Lengend Snippet: Flow cytometric analysis of HSC from secondary recipients of muscle cells. Bone marrow prepared from the secondary transplant recipient mouse 5 (Fig. ​(Fig.3)3) was stained with Hoechst 33342, anti-Ly-5.1-biotin, and Str-PE. (A and B) Whole bone marrow. (C and D) HSC. Whole bone marrow contained 0.03% HSC (determined by Hoechst dye efflux; refs. 21 and 22) and contained 63% Ly-5.1+ cells (B). The HSC population (C) contained 65% Ly-5.1+ cells (D).

Article Snippet: The nucleated cells were then stained with anti-Ly-5.1-biotin (clone A20), rat-IgG2a-FITC (R35–95), rat-IgG2b-FITC (A95–1), B220-FITC (RA3–6B2), Thy-1-FITC (30-H12), Gr-1-FITC (RB6–8C5), and Mac-1-FITC (M1/70) (all from PharMingen).

Techniques: Staining