Journal: Frontiers in Pharmacology

Article Title: The Inhibitory Effect of Curcumin Derivative J147 on Melanogenesis and Melanosome Transport by Facilitating ERK-Mediated MITF Degradation

doi: 10.3389/fphar.2021.783730

Figure Lengend Snippet: Effect of MAPK pathway on J147-induced hypomelanogenesis. (A) B16F10 cells were treated with J147 (4 μM) for the indicated time period (0–120 min), and the phosphorylation of the MEK, ERK, p38 and JNK were measured by western blot. (B) B16F10 was pretreated or not with 10 μM SB203580 (the inhibitor of p38) for 1 h before J147 was applied for 48 h. Melanin contents and the expression of Myosin Va, tyrosinase, MITF, Cdc42 and Rab27a were measured as described in methods. (C) B16F10 was pretreated or not with 10 μM PD98059 (the inhibitor of ERK) for 1 h before J147 was applied for 48 h. Melanin contents and the expression of Myosin Va, tyrosinase, MITF, Cdc42 and Rab27a were measured as described in methods. Data are expressed as the mean ± SE ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. non-treated cells. # p < 0.05 vs. J147-treated cells.

Article Snippet: We obtained antibodies against Myosin Va (sc-365986), KIF5b (sc-133184), GP100 (sc-393094), Cdc42 (sc-8401), Rab27a (sc-74586), p-JNK (sc-6254), JNK (sc-7345), p-p38 MAPK (sc-166182) and p38 MAPK (sc-398546) from Santa Cruz (CA, USA).

Techniques: Western Blot, Expressing

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Role of p38γ MAPK in Regulation of EMT and Cancer Stem Cells

doi: 10.1016/j.bbadis.2018.08.024

Figure Lengend Snippet: Effect of p38γ MAPK on EMT in breast cancer cells. MCF7 cells were stably transfected with either WT p38γ MAPK (MCF7-p38WT) or the active form of p38γ MAPK (MCF7-p38D179) plasmids as described in the Materials and Methods. A: MCF7 and MCF7-p38D179 cells were cultured on coverslips. The expression of E-cadherin was detected by immunofluorescent staining as described in the Materials and Methods. Arrows indicate the E-cadherin on the cell borders. Bar = 20 μM. B and D: Cell lysates of MCF7, MCF7-p38WT or MCF7-p38D179 were collected and the expression of EMT markers (E-cadherin and Vimentin), and p38γ MAPK was analyzed by immunoblotting. GAPDH served as a loading control. C: The expression of E-cadherin and p38γ MAPK was quantified and normalized to the expression of GAPDH. The experiment was replicated at least three time. * denote significant difference from MCF7 cells, p < 0.05. E: MCF7 cells stably expressing control shRNA (MCF7) and p38γ MAPK shRNA (MCF7-p38sh) were established as described in the Materials and Methods. The expression of E-cadherin, Vimentin, p38γ and p38α MAPK was examined with immunoblotting.

Article Snippet: Anti-Vimentin, p38α and p38γ antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Stable Transfection, Transfection, Cell Culture, Expressing, Staining, Western Blot, shRNA

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Role of p38γ MAPK in Regulation of EMT and Cancer Stem Cells

doi: 10.1016/j.bbadis.2018.08.024

Figure Lengend Snippet: Effect of p38γ MAPK on EMT in MCF7 cells over-expressing ErbB2. MCF7 cells over-expressing ErbB2 were stably transfected with control shRNA (MCF7-ErbB2) and p38γ MAPK shRNA (MCF7-ErbB2 p38sh) as described in the Methods. A: Cells were cultured on coverslips. The expression of E-cadherin was detected by immunofluorescent staining as described under the Materials and Methods. Arrows indicate the E-cadherin on the cell borders. Bar = 20 μM. B: Cell lysates of MCF7-ErbB2 and MCF7-ErbB2 p38sh cells were collected and the expression of EMT markers (Ecadherin and Vimentin), p38γ MAPK was analyzed by immunoblotting. GAPDH served as a loading control. C: The expression of E-cadherin and p38γ MAPK was quantified and normalized to the expression of GAPDH. The experiment was replicated at least three time. * denote significant difference from MCF7-ErbB2 cells, p < 0.05. D: The mRNA levels of E-cadherin, Vimentin, and p38γ MAPK in MCF7, MCF7-p38D179, MCF7-ErbB2 and MCF7-ErbB2 p38sh cells were examined by RT-PCR. The mRNA level of GAPDH was used as a loading control.

Article Snippet: Anti-Vimentin, p38α and p38γ antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Expressing, Stable Transfection, Transfection, shRNA, Cell Culture, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Role of p38γ MAPK in Regulation of EMT and Cancer Stem Cells

doi: 10.1016/j.bbadis.2018.08.024

Figure Lengend Snippet: Effect of p38γ MAPK on EMT in BT474 breast cancer cells. BT474 breast cancer cells were stably transfected with control shRNA (BT474) and p38γ shRNA (BT474 p38sh). A: The expression of E-cadherin was detected by immunofluorescent staining. Arrows indicate the E-cadherin on the cell borders. Bar = 20 μM. B: Cell lysates of BT474 and BT474 p38sh cells were collected and the expression of Ecadherin, Vimentin, p38γ, p38α MAPK, and GAPDH was analyzed by immunoblotting. C: The expression of E-cadherin and p38γ MAPK was quantified and normalized to the expression of GAPDH. The experiment was replicated at least three time. * denote significant difference from BT474 cells, p < 0.05. D: The mRNA levels of E-cadherin, Vimentin, and p38γ MAPK in BT474 and BT474 p38sh cells were analyzed by RT-PCR. The mRNA level of GAPDH was used as a loading control.

Article Snippet: Anti-Vimentin, p38α and p38γ antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Stable Transfection, Transfection, shRNA, Expressing, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Role of p38γ MAPK in Regulation of EMT and Cancer Stem Cells

doi: 10.1016/j.bbadis.2018.08.024

Figure Lengend Snippet: Effect of p38γ MAPK on CSCs and tumorsphere formation in breast cancer cells. A: MCF7 and MCF7-p38D179 cells were processed for ALDEFLUOR assay, followed by flow cytometry for the detection of CSCs. CSC population was calculated as percentage of total cells population. Each data point was mean ± SEM of three independent experiments. * p<0.05, denotes significant difference from the control groups. B: Cells (1,000 cells) were cultured on ultra-low attachment plates for assaying tumorsphere formation as described in the Materials and Methods. The number of tumorspheres was counted and calculated relative to MCF7 cells. Each data point was the mean ± SEM of three independent experiments. * p<0.05, denotes significant difference from MCF7 cells. C and D: MCF7-ErbB2 and MCF7-ErbB2 p38sh cells were analyzed for CSCs and tumorsphere formation as described above. * p < 0.05, denotes significant difference from MCF7- ErbB2 cells. E and F: BT474 and BT474 p38sh cells were analyzed for CSCs and tumorsphere formation as described above. * p < 0.05, denotes significant difference from BT474 cells.

Article Snippet: Anti-Vimentin, p38α and p38γ antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Flow Cytometry, Cell Culture

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Role of p38γ MAPK in Regulation of EMT and Cancer Stem Cells

doi: 10.1016/j.bbadis.2018.08.024

Figure Lengend Snippet: p38γ MAPK and miR-200b in their regulation of E-cadherin. A: The expression of miR-200b in MCF7, and MCF7-p38D179, MCF7-ErbB2 and MCF7-ErbB2 p38sh cells was analyzed by real-time PCR as described in the Materials and Methods. Each data point was the mean ± SEM of three independent experiments. p< 0.05, * denotes significant difference from MCF7 cells. # denotes significant difference from MCF7-ErbB2 p38shCon Si. B: MCF7-ErbB2 and MCF7-ErbB2 p38sh cells were treated with either controls or mimics/inhibitors for miR-200b or miR-34c (con mim, con inhi,200b mim, 34c mim, 200b inhi or 34c inhi) as described in the Materials and Methods. After 48 hours, E-cadherin levels were analyzed by immunoblotting. C: The expression of E-cadherin was quantified and normalized to the expression of GAPDH. The experiment was replicated at least three time. * denote significant difference from con min or con inhi, p < 0.05. D: MCF7-ErbB2 and MCF7-ErbB2 p38sh cells were treated with miR-200b mimics or inhibitor for 48 hours, then the expression of E-cadherin was detected by immunofluorescent staining. Arrows indicate the E-cadherin on the cell borders. Bar = 20 μM. E: The protein levels of p38γ MAPK, GATA3, and Suz12 were determined by immunoblotting. F: The relative miR-200b levels were determined by real-time PCR. G: MCF7 or MCF7-p38D179 cells were treated with either miRNA-200b inhibitor (200b inhi) or mimics (200b mim) for 48 hours. The expression of E-cadherin, p38γ MAPK, GATA3, Suz12, and GAPDH was examined by immunoblotting. The experiments were replicated three times. H: The relative expression of miRNA-200b was determined by real-time PCR. Each data point was the mean ± SEM of three independent experiments *p < 0.05, denotes significant difference from respective controls.

Article Snippet: Anti-Vimentin, p38α and p38γ antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Role of p38γ MAPK in Regulation of EMT and Cancer Stem Cells

doi: 10.1016/j.bbadis.2018.08.024

Figure Lengend Snippet: Effect of GATA3 on the expression of E-cadherin and miRNA-200b in breast cancer cells. MCF7 or MCF7-ErbB2 p38sh cells were transiently transfected with GATA3 siRNA for 48 hours. MCF7-p38D179 or MCF7-ErbB2 cells were transfected with GATA3 plasmid for 48 hours. A: The relative levels of miR-200b were determined by real-time PCR. Each data point was the mean ± SEM of three independent experiments. p< 0.05, * denotes significant difference from match controls. # denotes significant difference from MCF7 Con Si. δ is significant difference from MCF7-ErbB2 Con P. B and C: The expression of E-cadherin, GATA3, p38γ MAPK, Suz12, and GAPDH in MCF7, MCF7-p38D179, MCF7-ErbB2, and MCF7-ErbB2 p38sh cells transfected with either GATA3 siRNA or GATA3 plasmid was examined by immunoblotting analysis. D and E: The expression of E-cadherin was examined by immunofluorescent staining. Arrows indicated E-cadherin on the cell borders. Arrows indicate the E-cadherin on the cell borders. Bar = 20 μM. The experiments were replicated three times.

Article Snippet: Anti-Vimentin, p38α and p38γ antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Staining

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Role of p38γ MAPK in Regulation of EMT and Cancer Stem Cells

doi: 10.1016/j.bbadis.2018.08.024

Figure Lengend Snippet: Effect of p38γ MAPK GATA3 expression. A: mRNA levels of GATA3 in MCF7, MCF7-p38D179, MCF7-ErbB2 and MCF7-ErbB2 p38sh cells were determined by PCR. B: Changes in the protein levels of GATA3 over a course of time after cycloheximide (50 μg/ml) treatments were determined by immunoblotting. *denotes significant difference from 0 hours. C and D: The cell lysates from MCF7, MCF7-p38D179, MCF7 p38sh, MCF7-ErbB2, and MCF7-ErbB2 p38sh were immunoprecipitated with an antiubiquitin antibody, and then analyzed for GATA3 expression by immunoblotting. E and G: MCF7-p38D179 and MCF7-ErbB2 cells treated with either lysosome inhibitor (chloroquine, 100 μM)) or proteasome inhibitor (MG132, 10 μM) for 6 hours. The expression of GATA3 was then analyzed by immunoblotting. F and H: The expression of GATA3 was quantified and normalized to the expression of GAPDH. The experiment was replicated at least three time. * denote significant difference from respective controls, p < 0.05.

Article Snippet: Anti-Vimentin, p38α and p38γ antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Expressing, Western Blot, Immunoprecipitation

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Role of p38γ MAPK in Regulation of EMT and Cancer Stem Cells

doi: 10.1016/j.bbadis.2018.08.024

Figure Lengend Snippet: Signaling pathway for p38γ MAPK-promoted EMT. p38γ MAPK is activated by its upstream receptor kinases, such as ErbB2 and ErbB4 [4, 5, 57]. The activation of p38γ MAPK destabilizes GATA3 by ubiquitin-proteasome-dependent degradation. Decreased levels of GATA3 causes the down-regulation of miR-200b, a negative regulator of Suz12. Suz12 is a promoter of EMT and CSC in breast cancer cells; therefore, down-regulation of miR-200b may promote EMT and CSC through the induction of Suz12. Alternatively, GATA3 may promote EMT and CSC through other miRNAs or unknown mechanisms (dotted lines)[48–50]. miR-200b could also promote EMT in breast cancer cells through mechanisms independent of Suz12 (dotted lines) [46].

Article Snippet: Anti-Vimentin, p38α and p38γ antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: p38γ and p38δ Are Involved in T Lymphocyte Development

doi: 10.3389/fimmu.2018.00065

Figure Lengend Snippet: p38γ and p38δ deficiency decreases thymus cell number. (A) Lysates from WT, p38γ −/− , p38δ −/− , and p38γ/δ −/− thymocytes were immunoblotted with anti-total p38α, -p38β, -p38γ, and -p38δ antibodies, and with anti-α-tubulin. (B) Expression of p38 MAPK mRNA analysed by qPCR method from WT, p38γ −/− , p38δ −/− , and p38γ/δ −/− mouse thymus. Data show mean ± SD of triplicates from one representative experiment. Data were normalised to GAPDH mRNA. (C) Total cell numbers from 4-week-old (1-month) WT ( n = 17), p38γ −/− ( n = 15), p38δ −/− ( n = 15), and p38γ/δ −/− ( n = 16) mice, determined by counting isolated cell suspensions. * p ≤ 0.05; ** p ≤ 0.001.

Article Snippet: Anti-total p38α and anti-α-tubulin antibodies were from Santa Cruz, -p38β from Zymed, and -p38γ and -p38δ antibodies were produced and purified as described ( , ).

Techniques: Expressing, Isolation

Journal: Frontiers in Immunology

Article Title: p38γ and p38δ Are Involved in T Lymphocyte Development

doi: 10.3389/fimmu.2018.00065

Figure Lengend Snippet: Effect of p38γ and/or p38δ deletion in the development of double-negative (DN) T cell. Thymocytes from 1-month mice were stained with anti-CD3, -CD4, -CD8, -CD44, and -CD25 antibodies and positive cells were analysed by flow cytometry. (A) Representative flow cytometry profiles are shown. (B,C) CD4 − CD8 − thymocytes from 1-month mice were analysed for CD44 and CD25 expression after simultaneous staining with anti-CD44 and -CD25 antibodies. The percentages (B) and total numbers (C) of DN1, DN2, DN3, and DN4 cells were determined. Each dot represents a single mouse. * p ≤ 0.05; ** p ≤ 0.001. (D) Ratio of DN2/DN1, DN3/DN2, and DN4/DN3 cell number. * p ≤ 0.05; ** p ≤ 0.001; *** p ≤ 0.0001.

Article Snippet: Anti-total p38α and anti-α-tubulin antibodies were from Santa Cruz, -p38β from Zymed, and -p38γ and -p38δ antibodies were produced and purified as described ( , ).

Techniques: Staining, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: p38γ and p38δ Are Involved in T Lymphocyte Development

doi: 10.3389/fimmu.2018.00065

Figure Lengend Snippet: p38γ and p38δ modulate lymphoid cell development in thymus. (A) Thymocytes from 1-month mice were stained with anti-CD3, -CD4, and -CD8 antibodies and the percentage of positive cells was analysed by flow cytometry. Representative flow cytometry profiles are shown. (B,C) Thymocytes from 1-month mice were stained simultaneously with anti-CD4 and -CD8 antibodies, and the percentages (B) and total number (C) of double-negative (DN), CD4 + , CD8 + , and double-positive (DP) cells were analysed by flow cytometry. Each dot represents a single mouse. * p ≤ 0.05; ** p ≤ 0.001. (D) Table showing the average cell number and the SD of thymocyte subpopulations from mice represented in panel (C) .

Article Snippet: Anti-total p38α and anti-α-tubulin antibodies were from Santa Cruz, -p38β from Zymed, and -p38γ and -p38δ antibodies were produced and purified as described ( , ).

Techniques: Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: p38γ and p38δ Are Involved in T Lymphocyte Development

doi: 10.3389/fimmu.2018.00065

Figure Lengend Snippet: Schematic representation indicating the different stages of T cell development partially controlled by p38γ and/or p38δ.

Article Snippet: Anti-total p38α and anti-α-tubulin antibodies were from Santa Cruz, -p38β from Zymed, and -p38γ and -p38δ antibodies were produced and purified as described ( , ).

Techniques:

Journal: Frontiers in Immunology

Article Title: p38γ and p38δ Are Involved in T Lymphocyte Development

doi: 10.3389/fimmu.2018.00065

Figure Lengend Snippet: Effect of kinase-inactive p38γ (D171A) in late T cell development. (A) Genomic DNA purified from tail biopsy sample was used as a template for PCR as in Ref. . (B) Lysates from WT and p38γ 171A/171A thymocytes were immunoblotted with anti-total p38α, -p38γ, and -p38δ antibodies. (C) Total cell numbers from 4-week-old (1-month) WT and p38γ 171A/171A mice, determined by counting isolated cell suspensions. Thymocytes from 1-month WT and p38γ 171A/171A mice were stained simultaneously with anti-CD4 and -CD8 antibodies, and the percentages (D) and total number (E) of double-negative (DN), CD4 + , CD8 + , and double-positive (DP) cells were analysed by flow cytometry as in Figure . p38γ −/− mice are included in panel (D) as control. Each dot represents a single mouse. * p ≤ 0.05; ** p ≤ 0.001; *** p ≤ 0.0001.

Article Snippet: Anti-total p38α and anti-α-tubulin antibodies were from Santa Cruz, -p38β from Zymed, and -p38γ and -p38δ antibodies were produced and purified as described ( , ).

Techniques: Purification, Isolation, Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: p38γ and p38δ Are Involved in T Lymphocyte Development

doi: 10.3389/fimmu.2018.00065

Figure Lengend Snippet: Effect of p38γ and/or p38δ deletion in the generation of γδTCR- and αβTCR-expressing T cells. Thymocytes from 1-month mice were stained with anti-CD3, -CD4, -CD8, -CD27, and -TCRγδ or -TCRαβ antibodies and cells were analysed by flow cytometry. (A) Analysis of CD27 expression in DN3 thymocytes. Data are representative of five different staining. (B) The percentages of CD3 + , CD4 − , CD8 − , and -TCRγδ + cells were determined. (C) Representative flow cytometry profiles are shown. Numbers indicate the percentage of cells falling into the respective regions. (D) The percentages of the different T cell populations were determined. Each dot represents a single mouse. * p ≤ 0.05; ** p ≤ 0.001. (E) Mean fluorescence intensity (MFI) levels in αβTCR CD8 + cells. Data show mean ± SD from one representative experiment with five mice.

Article Snippet: Anti-total p38α and anti-α-tubulin antibodies were from Santa Cruz, -p38β from Zymed, and -p38γ and -p38δ antibodies were produced and purified as described ( , ).

Techniques: Expressing, Staining, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: p38γ and p38δ Are Involved in T Lymphocyte Development

doi: 10.3389/fimmu.2018.00065

Figure Lengend Snippet: Characterisation of lymph nodes (LNs) in p38γ/δ-deficient mice. (A) LNs lysates from WT mice were immunoblotted with anti-total p38α, -p38β, -p38γ, and -p38δ antibodies. Results from two independent samples are shown. (B) Representative popliteal (top) and inguinal (bottom) LN from adult WT and p38γ/δ −/− mice. Bar, 1 cm. (C) Total cell number in draining LNs of 4-week-old (1-month) of the indicated genotypes. Each dot represents a single mouse. ** p ≤ 0.001. (D–F) LN cells from 1-month WT, p38γ −/− , p38δ −/− , and p38γ/δ −/− mice were stained with anti-CD3, -CD4, and -CD8, and the percentage of the indicated populations was examined. (D) Representative flow cytometry profiles and dot plots. T cells were gated as CD3 + cells. (E,F) Graphs showing total number (F) and percentages (E) of LN T cell populations. Each dot represents a single mouse. * p ≤ 0.05; ** p ≤ 0.001.

Article Snippet: Anti-total p38α and anti-α-tubulin antibodies were from Santa Cruz, -p38β from Zymed, and -p38γ and -p38δ antibodies were produced and purified as described ( , ).

Techniques: Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: p38γ and p38δ Are Involved in T Lymphocyte Development

doi: 10.3389/fimmu.2018.00065

Figure Lengend Snippet: p38γ and p38δ are not required for lymphoid and myeloid cells development in lymph node (LN).

Article Snippet: Anti-total p38α and anti-α-tubulin antibodies were from Santa Cruz, -p38β from Zymed, and -p38γ and -p38δ antibodies were produced and purified as described ( , ).

Techniques:

Journal: Oncotarget

Article Title: Chronic ethanol exposure enhances the aggressiveness of breast cancer: the role of p38γ

doi: 10.18632/oncotarget.6508

Figure Lengend Snippet: MCF7 cells were exposed to ethanol (0 or 100 mg/dl) for a month, then xenografted into nude mice as described in the Experimental procedures. A. Ethanol-pretreated cells ranging from 10 3 to 10 6 cells/100 μl in PBS were subcutaneously inoculated into the lower flank of nude mice ( n = 6). One month after inoculation, tumorigenicity was evaluated and presented as percentage of the original inoculation. * p < 0.05. B. Tumor sizewas measured weekly and tumor volume (mm 3 ) was calculated as described in the Experimental procedures. * p < 0.05. C. Tumor tissues from control or ethanol-exposed groups were fixed, sectioned and processed for IHC staining of phospho-p38γ (p-p38γ).

Article Snippet: Anti-p38α, p38β, p38γ MAPK and RhoC antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Immunohistochemistry

Journal: Oncotarget

Article Title: Chronic ethanol exposure enhances the aggressiveness of breast cancer: the role of p38γ

doi: 10.18632/oncotarget.6508

Figure Lengend Snippet: MCF7 cells were exposed to ethanol (0 or 100 mg/dl) for 10 days, 1 month or 2 months. A. Cell lysates were collected and then equal amount of proteins were immuoprecipitated (IP) with an anti-p38γ MAPK antibody and then immunoblotted (IB) with an antibody directed against pan phosphorylated p38 MAPK (p-p38). B. Proteins were IP with an anti p-p38 antibody and then IB with an anti-p38α antibody. C. MCF7 cells were exposed to ethanol (0, 100, 200 or 400 mg/dl) for indicated times, then proteins were collected and IP with an anti-p-p38 antibody and IB with an anti-p38γ MAPK antibody. D. MCF7 cells were exposed to ethanol (100 mg/dl) for 0.5–12 hours. The expression of phosphorylated p38γ MAPK, total p38γ MAPK and RhoC was determined by immunoblotting. E. Equal amount of proteins were IP with p38γ or p38α, and then IB with either a commercial anti-pan phosphorylated p38 antibody (p-p38) or a specific anti-phosphorylated-p38γ antibody (p-p38γ) (21st Century Biochemical, please see Materials and Methods). F. The same protein samples described on panel A was analyzed with immunoblotting using the specific anti-p-p38γ MAPK antibody. G. The expression of phosphorylated p38γ in MCF7 and SP-MCF7 cells was evaluated using a specific anti-p-p38γ antibody as described above. All experiments were replicated at least three times.

Article Snippet: Anti-p38α, p38β, p38γ MAPK and RhoC antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: Chronic ethanol exposure enhances the aggressiveness of breast cancer: the role of p38γ

doi: 10.18632/oncotarget.6508

Figure Lengend Snippet: We have established MCF7 cells stably expressing control shRNA (Consh) and p38γ shRNA (p38γsh) as described in the Materials and Methods. The expression of p38γ and p38α was examined with immunoblotting A. These cells were exposed to ethanol (100 mg/dl) for 10 days then assayed for cell scattering in a 3-D culture system B. and anchorage-independent colonies formation C. The results were presented relative to the controls. Each data point was the mean ± SEM of three experiments. * denotes significant difference from control groups. # denotes significant difference from p38γ shRNA EtOH groups.

Article Snippet: Anti-p38α, p38β, p38γ MAPK and RhoC antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Stable Transfection, Expressing, shRNA, Western Blot

Journal: Oncotarget

Article Title: Chronic ethanol exposure enhances the aggressiveness of breast cancer: the role of p38γ

doi: 10.18632/oncotarget.6508

Figure Lengend Snippet: MCF7 cells stably expressing control shRNA (Consh) and p38γ shRNA (p38γsh) were exposed to ethanol (0 or 100 mg/dl) for 10 days. After that, cell migration A. invasion B. cancer stem-like cell population C. and cell viability D. were evaluated as described in the Materials and Methods. The results were expressed relative to the controls. Each data point was the mean ± SEM of three experiments. * denotes significant difference from control groups. # denotes significant difference from ethanol-treated p38γsh groups.

Article Snippet: Anti-p38α, p38β, p38γ MAPK and RhoC antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Stable Transfection, Expressing, shRNA, Migration

Journal: Oncotarget

Article Title: Chronic ethanol exposure enhances the aggressiveness of breast cancer: the role of p38γ

doi: 10.18632/oncotarget.6508

Figure Lengend Snippet: MCF7 cells stably expressing control shRNA and shRNA for p38γ MAPK (p38γsh) were exposed to ethanol (0 or 100 mg/dl) for a month, then 5 × 10 6 cells (in 100 ul PBS) were inoculated into nude mice on both sides of the lower flank as described in the Materials and Methods. A. Four weeks after inoculation, the tumors were measured and the average volume was calculated. * denotes significant difference from mice inoculated with MCF7 cells. # denotes significant difference from mice inoculated with MCF7 cells expressing p38γ shRNA. B. At completion of experiments, mice were sacrificed and analyzed for tumor metastasis as described in the Materials and Methods. C. Lung tissues were fixed, sectioned and stained. The image shows metastatic carcinomas in the lungs of mice that were inoculated with ethanol-treated MCF7 cells.

Article Snippet: Anti-p38α, p38β, p38γ MAPK and RhoC antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Stable Transfection, Expressing, shRNA, Staining

Journal: Oncotarget

Article Title: Chronic ethanol exposure enhances the aggressiveness of breast cancer: the role of p38γ

doi: 10.18632/oncotarget.6508

Figure Lengend Snippet: A. MCF7 cells were exposed to ethanol (0, 100, 200 or 400 mg/dl) for 10 days, 1 month or 2 months, then the expression of RhoC was examined by immunoblotting. B. MCF7 cells stably expressing control shRNA and p38γ shRNA (p38γsh) were exposed to ethanol (0 or 100 mg/dl) for 10 days. After that, cell lysates were IP with an anti-p38γ antibody, and then IB with an anti p-p38 MAPK antibody. The expression of RhoC was examined. C. Cell lysates were IP with an anti-ubiquitin antibody and IB with an anti-RhoC antibody. D. MCF7 cells were exposed to ethanol (0 or 100 mg/dl) for 10 days, and then treated with either control siRNA or RhoC siRNA for 48 hours. After that, cells were assayed for the migration during a 12-hour-period with/without ethanol (100 mg/dl). E. Cell invasion was evaluated. Each data point was the mean ± SEM of three experiments and presented relative to the controls values. * denotes significant difference from the controls. # denotes significant difference from long term EtOH (LT EtOH). δ denotes significant difference from corresponding controls.

Article Snippet: Anti-p38α, p38β, p38γ MAPK and RhoC antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA).

Techniques: Expressing, Western Blot, Stable Transfection, shRNA, Migration

Journal: Oncotarget

Article Title: Combined deletion of p38γ and p38δ reduces skin inflammation and protects from carcinogenesis

doi:

Figure Lengend Snippet: A. Number of tumours per mouse and percentage of mice with tumours are shown at indicated times. WT ( n = 9), p38γ −/− ( n = 15), p38δ −/− ( n = 16) and p38γ/δ −/− ( n = 9) mice were treated with DMBA/TPA (see Methods) and the skin was monitored for tumour growth at indicated times. Tumour number per mouse shown as mean ± SEM. ns, not significant; * p ≤0.05; ** p ≤ 0.01; *** p ≤ 0.001 relative to WT mice or between indicated genotypes (black lines). B. Papilloma (P) and healthy skin (H) protein extracts from two WT mice (WT1 and WT2) were immunoblotted with antibodies to active phosphorylated p38 (P-p38α, P-p38δ) (green), and total p38δ (red) and p38γ. Blots were analysed using the Odyssey infrared imaging system. P-p38δ was visualised in yellow when colours were merged. Results were similar in three independent experiments. C. Expression of p38γ, p38δ and p38α protein and mRNA. WT, p38γ −/− and p38δ −/− papilloma protein extracts were immunoblotted with the indicated antibodies. Representative blots are shown. Band intensities from the p38γ and p38α immunoblot were quantified using the Odyssey infrared imaging system. Quantification is represented as p38γ/p38α. Data show mean ± SEM. qPCR of p38MAPK mRNA in total RNA from WT, p38γ −/− or p38δ −/− papilloma and in total RNA from healthy skin (H) from WT mice. Expression of the different p38 mRNA was normalised to GAPDH. Data show mean ± SEM from one representative experiment of at least three with similar results. D. Representative H&E-stained sections of skin tumour at week 29 (Panel A). Scale bars: 500 μm. E. Histograms of tumour size distribution in A. at indicated times. F. Proliferation in tumours from WT, p38γ −/− and p38δ −/− mice (at week 29) was evaluated by BrdU staining. BrdU positive cells (red) were counted and represented as percentage of total basal keratinocytes. Nuclei are Hoechst33342-stained (blue). Results show mean ± SEM ( n = 3-6 tumours/group). ns, not significant. Scale bars: 100 μm. G. Papilloma sections were stained to evaluate P-STAT3. Scale bars: 50 μm. Representative sections are shown. (See Materials and Methods).

Article Snippet: Anti-STAT3 and -p38α were from Santa Cruz, anti-active phospho-JNK1/2 (Thr183-Tyr185; P-JNK) from Biosource, anti-BrdU and -myeloperoxidase (MPO) from Abcam, and anti-p38γ and -p38δ antibodies were raised and purified as described [ , ].

Techniques: Imaging, Expressing, Western Blot, Staining, BrdU Staining

Journal: Oncotarget

Article Title: Combined deletion of p38γ and p38δ reduces skin inflammation and protects from carcinogenesis

doi:

Figure Lengend Snippet: A. shControl-, shp38γ-, shp38δ- and shp38γ/δ-A431 cell extracts (50 μg) were examined by immunoblotting with the indicated antibodies to determine p38γ and p38δ expression. B. Immunodeficient nude mice received subcutaneous injections of shControl-A431 cells, shp38γ-A431 cells, shp38δ-A431 cells and shp38γ/δ-A431 cells, and tumour volume was measured periodically as indicated. Values are means ± SD for 12 mice. Inset: Representative photographs of mouse tumour at day 19. Arrows indicate injection sites.

Article Snippet: Anti-STAT3 and -p38α were from Santa Cruz, anti-active phospho-JNK1/2 (Thr183-Tyr185; P-JNK) from Biosource, anti-BrdU and -myeloperoxidase (MPO) from Abcam, and anti-p38γ and -p38δ antibodies were raised and purified as described [ , ].

Techniques: Western Blot, Expressing, Injection

Journal: Oncotarget

Article Title: Combined deletion of p38γ and p38δ reduces skin inflammation and protects from carcinogenesis

doi:

Figure Lengend Snippet: A. p38γ and p38δ expression in the skin and in keratinocytes. qPCR of p38MAPK mRNA in total RNA from WT, p38γ −/− or p38δ −/− skin and in total RNA from keratinocytes from WT mice. Expression of the different p38 mRNA was normalised to GAPDH. Data show mean ± SEM ( n = 3 mice/group). B. WT, p38γ −/− and p38δ −/− skin extracts and WT keratinocytes lysates (50 μg) were immunoblotted with antibodies to total p38γ, p38δ and p38α. Representative blots are shown. C. WT and p38γ/δ −/− mice were treated for 24 h with DMBA or acetone as control. Skin sections were immunofluorescence-stained to evaluate DMBA-induced DNA damage response (γH2AX). γH2AX + cells (red) were quantified; at least 8 fields/mouse were scored. Results show mean ± SEM ( n = 4 mice/group). ns, not significant, relative to WT mice in the same conditions. Scale bars: 100 μm. D. , E. Apoptosis in mouse skin was evaluated by TUNEL staining (red) at 24 h post-DMBA application. Apoptotic cells were counted; 12 fields/mouse were scored. Results show mean ± SEM ( n = 4 mice/group). ns, not significant, relative to WT mice. Panel D. shows quantitation of follicular apoptotic cells and E. shows interfollicular apoptotic cells. Scale bars: 50 μm. In C. and D. nuclei are Hoechst33342-stained (blue).

Article Snippet: Anti-STAT3 and -p38α were from Santa Cruz, anti-active phospho-JNK1/2 (Thr183-Tyr185; P-JNK) from Biosource, anti-BrdU and -myeloperoxidase (MPO) from Abcam, and anti-p38γ and -p38δ antibodies were raised and purified as described [ , ].

Techniques: Expressing, Immunofluorescence, Staining, TUNEL Assay, Quantitation Assay

Journal: Oncotarget

Article Title: Combined deletion of p38γ and p38δ reduces skin inflammation and protects from carcinogenesis

doi:

Figure Lengend Snippet: A. Representative H&E staining of skin sections from WT, p38γ −/− , p38δ −/− and p38γ/δ −/− mice treated for 31 h with TPA or acetone as control. Epidermal thickness, indicated by black lines, was measured. Results show mean ± SEM ( n = 4 mice/group), * p ≤ 0.05; ** p ≤ 0.01; *** p < 0.001. Scale bars: 100 μm. B. Proliferation in skin of WT, p38γ −/− , p38δ −/− and p38γ/δ −/− mice was evaluated by BrdU staining at 31 h post-TPA application. BrdU positive cells (red) were counted; 12 fields/mouse were usually scored. Nuclei are Hoechst33342-stained (blue). Results show mean ± SEM ( n = 4 mice/group). * p ≤ 0.05; ** p < 0.01, relative to WT mice. Scale bars: 50 μm.

Article Snippet: Anti-STAT3 and -p38α were from Santa Cruz, anti-active phospho-JNK1/2 (Thr183-Tyr185; P-JNK) from Biosource, anti-BrdU and -myeloperoxidase (MPO) from Abcam, and anti-p38γ and -p38δ antibodies were raised and purified as described [ , ].

Techniques: Staining, BrdU Staining

Journal: Oncotarget

Article Title: Combined deletion of p38γ and p38δ reduces skin inflammation and protects from carcinogenesis

doi:

Figure Lengend Snippet: A. Skin extracts (50 μg) from WT and p38γ/δ −/− mice, treated with acetone (control, time 0) or with TPA for the indicated times, were immunoblotted with antibodies to active phosphorylated p38 (P-p38α, P-p38γ) and total p38α, p38γ and p38δ. B. WT mice were treated with TPA for 120 min as in A. . Endogenous p38δ and p38γ were immunoprecipitated from WT skin extracts (2 mg). Pellets were immunoblotted with anti-P-p38 (P-p38δ, P-p38γ) or -p38δ and -p38γ antibodies. Representative blots are shown. C. Skin protein extracts (50 μg) from WT, p38γ −/− and p38δ −/− treated with TPA for the indicated times, were immunoblotted with antibodies to active phosphorylated p38 (P-p38α, P-p38γ), and total p38α, p38γ and p38δ. Results were similar in three independent experiments. D. p38γ −/− mice were treated with TPA for 120 min as in C. . Endogenous p38δ was immunoprecipitated from skin extracts (2 mg). Pellets were immunoblotted with anti-P-p38 (P-p38δ) or -p38δ antibodies. Representative blots are shown. E. Skin protein extracts (50 μg) from WT and p38δ −/− mice, treated with TPA for the indicated times, were immunoblotted with antibodies as in C. . Bands from the immunoblots were quantified using the Odyssey infrared imaging system. Quantification is represented as P-p38γ/p38γ. Data show mean ± SEM. F. WT and p38γ/δ −/− mouse skin were treated as in A. . Skin extracts (50 μg) were immunoblotted with antibodies to active phosphorylated ERK1/2 (P-ERK1/2), active phosphorylated JNK1/2 (P-JNK1/2) or phosphorylated NF-κB-p105. Total protein levels of ERK1/2, JNK1/2, and NF-κB-p105 were also measured in the same lysates as loading controls. G. Skin extracts (50 μg) from control and TPA-treated WT and p38γ/δ −/− mice were immunoblotted with antibodies to phospho- and total STAT3. Representative blots are shown. Bands were quantified using the Odyssey infrared imaging system. Quantification is represented as densities ratio P-STAT3 /STAT3. * p ≤0.05.

Article Snippet: Anti-STAT3 and -p38α were from Santa Cruz, anti-active phospho-JNK1/2 (Thr183-Tyr185; P-JNK) from Biosource, anti-BrdU and -myeloperoxidase (MPO) from Abcam, and anti-p38γ and -p38δ antibodies were raised and purified as described [ , ].

Techniques: Immunoprecipitation, Western Blot, Imaging

Journal: Oncotarget

Article Title: Combined deletion of p38γ and p38δ reduces skin inflammation and protects from carcinogenesis

doi:

Figure Lengend Snippet: A. Relative mRNA expression of indicated genes at different times was determined by qPCR in TPA-treated WT and p38γ/δ −/− mouse skin and normalised to GAPDH mRNA. Data show mean ± SEM ( n = 3-6). ns, not significant; * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001, relative to WT mice in the same conditions. B. Skin protein extracts from three WT and three p38γ/δ −/− mice (300 μg total), treated with TPA for 8 (top) and 24 h (bottom), were mixed with an antibody mixture and incubated with the Mouse Cytokine Array Panel A membrane as indicated by the manufacturer (R&D Systems). Pixel densities on the film were analysed using ImageJ software. ns, not significant; * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001. C. Relative mRNA expression was determined by qPCR for indicated genes in TPA-treated WT and p38γ/δ −/− keratinocytes and normalised to GAPDH mRNA. In panel B. and C. data show mean ± SEM ( n = 3). ns, not significant; * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001, relative to WT in the same conditions.

Article Snippet: Anti-STAT3 and -p38α were from Santa Cruz, anti-active phospho-JNK1/2 (Thr183-Tyr185; P-JNK) from Biosource, anti-BrdU and -myeloperoxidase (MPO) from Abcam, and anti-p38γ and -p38δ antibodies were raised and purified as described [ , ].

Techniques: Expressing, Incubation, Software

Journal: Oncotarget

Article Title: Combined deletion of p38γ and p38δ reduces skin inflammation and protects from carcinogenesis

doi:

Figure Lengend Snippet: A. - C. Skin cells from 24-h TPA-treated (or acetone as control) WT and p38γ/δ −/− mice were stained with anti-CD45, -CD3, -γδ TCR, -F4/80 and -Ly6G antibodies. A. Percentages of CD45 + cells are shown. CD45 + cells were gated and percentages of B. CD3 + , γδ TCR + and F4/80 + cells, or C. Ly6G + cells were analysed by flow cytometry. Data show mean ± SEM ( n = 3-4 per experiment and condition); ** p ≤ 0.01, *** p ≤ 0.001, ns, not significant. Representative profiles are shown in C. . (D, E) WT or p38γ/δ −/− mice were treated for 12 or 24 h D. and for 24 h E. with TPA or acetone as control. D. Skin sections were immunohistochemically stained to evaluate neutrophils (MPO). MPO + cells were quantified; 30 fields/mouse were usually scored. Results show mean ± SEM ( n = 3 mice/group). * p ≤ 0.05; ns, not significant; Scale bars: 20 μm. E. WT and p38γ/δ −/− skin lysates (50 μg) were immunoblotted with antibodies to MPO and ERK1/2 (loading control). Immunoblots were quantified using the Odyssey infrared imaging system; normalised MPO band densities are represented numerically below the blot.

Article Snippet: Anti-STAT3 and -p38α were from Santa Cruz, anti-active phospho-JNK1/2 (Thr183-Tyr185; P-JNK) from Biosource, anti-BrdU and -myeloperoxidase (MPO) from Abcam, and anti-p38γ and -p38δ antibodies were raised and purified as described [ , ].

Techniques: Staining, Flow Cytometry, Western Blot, Imaging