Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: RETRACTED: Blockade of TNF-α signaling suppresses the AREG-mediated IL-6 and IL-8 cytokines secretion induced by anti-Ro/SSA autoantibodies.

doi: 10.1038/labinvest.2010.168

Figure Lengend Snippet: Figure 1 Semiquantitative RT-PCR and real-time PCR for Furin, TACE and AREG genes expression. (a) RT-PCR analysis of Furin, TACE and AREG mRNA extracted from SGEC treated with anti-Ro/SSA autoantibodies. M, marker; control, untreated SGEC; HIgG, SGEC treated with IgG fractions extracted from sera of healthy donors; anti-Ro, SGEC treated with anti-Ro/SSA autoantibodies. RT-PCR of GADPH was used as control. Band intensities were analyzed by densitometry (b). (c) Real-time PCR for Furin, TACE and AREG genes expression. Representative histograms of mRNA levels of Furin, TACE and AREG in untreated SGEC (control), SGEC treated with healthy IgG (HIgG), anti-Ro/SSA autoantibodies (anti-Ro). The mRNA levels of the housekeeping gene, b-2 microglobulin, were quantified between untreated control cells and variously treated cells (data represent the mean±s.e. of five independent experiments).

Article Snippet: Membranes were incubated for 90 min with rabbit anti-human Furin polyclonal antibody (pAb), goat antihuman TACE pAb (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human AREG mAb (R&D Systems, Minneapolis, MN USA), and for 30 min with the relative secondary antibodies-HRP conjugates (Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Marker, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: RETRACTED: Blockade of TNF-α signaling suppresses the AREG-mediated IL-6 and IL-8 cytokines secretion induced by anti-Ro/SSA autoantibodies.

doi: 10.1038/labinvest.2010.168

Figure Lengend Snippet: Figure 2 Analysis of Furin, TACE and AREG expression in anti-Ro/SSA Abs-treated SGEC. (a) Flow cytometric analysis of Furin, TACE and AREG expression in SGEC after anti-Ro/SSA Abs treatment. Examples of flow cytometric images from one representative experiment. (A) Furin expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC; (B) intracellular active TACE expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC; (C) AREG expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC. (b) Western blot analysis of Furin, TACE and AREG proteins expression in SGEC treated or not with anti-Ro/SSA. Immunoblotting gave rise to bands of the expected size (97 kDa for Furin, 80 kDa for active TACE and 50 kDa for AREG). b-Actin was used as protein loading control. (c) Detection of soluble AREG by ELISA. Secreted AREG was detected by ELISA in the conditioned medium. Control, untreated SGEC; HIgG, SGEC treated with IgG fractions extracted from sera of healthy donors; anti-Ro, SGEC treated with anti-Ro/SSA autoantibodies. (Data represent the mean±s.e. of four independent experiments).

Article Snippet: Membranes were incubated for 90 min with rabbit anti-human Furin polyclonal antibody (pAb), goat antihuman TACE pAb (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human AREG mAb (R&D Systems, Minneapolis, MN USA), and for 30 min with the relative secondary antibodies-HRP conjugates (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences

Article Title: ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NFκB activation

doi: 10.1007/s00018-022-04186-5

Figure Lengend Snippet: Upregulation of the SASP by ROS and Padi2 knockdown leads to cellular senescence and the inhibition of the SASP ameliorates cellular senescence and osteogenic dysfunction. A Representative images of SA-β-Gal staining. MC3T3-E1 cells were cultured in the conditioned medium (CM) from MC3T3-E1 transfected with siCont or siPadi2 for 8 days and SA-β-Gal staining was performed. Percentage of SA-β-Gal-positive cells in each group was calculated as the percentage of SA-β-Gal-positive cells relative to the total cells stained with DAPI in the same field (8–10 fields/group). Magnification ×20, Scale bars 100 μm. Bars, statistical significance was determined using one-way ANOVA for multiple comparisons, * P < 0.05, *** P < 0.001. Three independent experiments were performed. B MC3T3-E1 cells were cultivated under Padi2 KD-CM for 8 days or siCont-CM, followed by performing RT-qPCR of Cdkn1a. Data for RT-qPCR are presented as the mean of three replicates; bars, ± SD. Statistical significance was determined using one-way ANOVA for multiple comparisons, ** P < 0.01, *** P < 0.001; n.s., not significant. Two independent experiments were performed. C Analysis of the relative expression levels of the SASP genes by RNA-seq data (black bar) and RT-qPCR (gray bar). Data are the fold change of 4 day-H 2 O 2 -treated group relative to the non-treated control. The red and blue-dotted lines mean fold change 1 and − 1, respectively. Data for RT-qPCR are presented as the mean of three replicates; bars, ± SD. statistical significance was determined using two-tailed Student’s t-test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n.s., not significant. Three independent experiments were performed for RT-qPCR. D MC3T3-E1 cells were transfected with siCont, siPadi2 #2 or siPadi2 #3 and then incubated for additional 4 days in the osteogenic medium. The expression level of each gene was measured by RT-qPCR and the fold change in mRNA levels was calculated by normalization to Gapdh . Data are presented as the mean of three replicates; bars, ± SD; statistical significance was determined using one-way ANOVA for multiple comparisons, * P < 0.05, *** P < 0.001, **** P < 0.0001. Three independent experiments were performed. E Five days-cell culture soup from MC3T3-E1 cells transfected with siCont, siPadi2 #2, or siPadi2 #3 was collected and the levels of CCL2, CCL5, and CCL7 in each cell culture soup were measured by ELISA. Data are presented as the mean of three replicates; bars, ± SD; statistical significance was determined using one-way ANOVA for multiple comparisons, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Three independent experiments were performed. F Representative images of SA-β-Gal staining. MC3T3-E1 cells knocked down with siCont or siPadi2 were treated with IgG or each neutralizing antibody for 4 days and SA-β-Gal staining was performed. Quantity of SA-β-Gal-positive cells was expressed as the percentage of SA-β-Gal-positive cells relative to the total cells stained with DAPI in the same field (8–10 fields/group). Magnification ×20, Scale bars 100 μm. bars, mean ± SD. statistical significance was determined using two-way ANOVA for multiple comparisons, *** P < 0.001. Three independent experiments were performed. G When MC3T3-E1 cells transfected with siCont, siCcl2, siCcl5, or siCcl7 were fully confluent, cells were treated with 100 μM H 2 O 2 for 4 days in osteogenic medium and ALP staining was performed. Quantification of ALP staining was performed by ImageJ. Bars, mean ± SD; statistical significance was determined using one-way or two-way ANOVA for multiple comparisons, * P < 0.05, ** P < 0.01, **** P < 0.0001; ns, not significant. H When MC3T3-E1 cells knocked down with siPadi2 in combination with or without siCcl2, 5, or 7 were fully confluent, cells were changed with osteogenic medium and incubated for 4 days and ALP staining was performed. Quantification of ALP staining (right). Bars, mean ± SD; statistical significance was determined using one-way or two-way ANOVA for multiple comparisons, * P < 0.05, ** P < 0.01,; ns not significant. Three independent experiments were performed

Article Snippet: Antibodies against the following proteins were used in this study: Padi2 (66386–1-Ig; Proteintech, Rosemont, IL, USA), α-tubulin (sc-8035; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), β-actin (sc-47778; Santa Cruz Biotechnology.), LaminA/C (sc-376248; Santa Cruz Biotechnology), p21 (#2947; Cell Signaling Technology, Inc., Danvers, MA, USA), mouse CCL2 antibody (AB-479-NA; R&D Systems, Inc, Minneapolis, MN, USA), mouse CCL5 antibody (AF478; R&D Systems), mouse CCL7 antibody (AF-456-NA; R&D Systems), and normal goat IgG control (AB-108-C; R&D Systems).

Techniques: Knockdown, Inhibition, Staining, Cell Culture, Transfection, Quantitative RT-PCR, Expressing, RNA Sequencing, Control, Two Tailed Test, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences

Article Title: ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NFκB activation

doi: 10.1007/s00018-022-04186-5

Figure Lengend Snippet: Schematic diagram depicted in the mechanism of oxidative stress-accelerated senescence and dysfunction of osteoblasts. The reduction of PADI2 by oxidative stress induces upregulation of CCL2, 5, and 7 known as the SASP, through the activation of NFκB signaling, leading to making a senescent environment and propagating cellular senescence to surrounding cells. Targeting these regulatory processes may be an effective way to prevent or treat excessive ROS-promoted cellular senescence and aging-related bone diseases

Article Snippet: Antibodies against the following proteins were used in this study: Padi2 (66386–1-Ig; Proteintech, Rosemont, IL, USA), α-tubulin (sc-8035; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), β-actin (sc-47778; Santa Cruz Biotechnology.), LaminA/C (sc-376248; Santa Cruz Biotechnology), p21 (#2947; Cell Signaling Technology, Inc., Danvers, MA, USA), mouse CCL2 antibody (AB-479-NA; R&D Systems, Inc, Minneapolis, MN, USA), mouse CCL5 antibody (AF478; R&D Systems), mouse CCL7 antibody (AF-456-NA; R&D Systems), and normal goat IgG control (AB-108-C; R&D Systems).

Techniques: Activation Assay

Journal: Development (Cambridge, England)

Article Title: Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate

doi: 10.1242/dev.134023

Figure Lengend Snippet: Ectopic IL6 family ligands result in accelerated AT2 differentiation via STAT3 activation. (A) Schematic and section of E15.5 slice culture resulting in differentiation of mature saccules with AT1 and AT2 cells in the presence of Dx. Green, pro-SFTPC; red, PDPN. (B) Schematic of IL6 and LIF experiments. Slices from individual lungs were split between two conditions for internal controls. (C,D) Sections from control, IL6- and LIF-exposed wild-type lungs. (C) Red, pSTAT3; white, E-CAD (epithelium). (D) Red, LAMP3 (differentiating AT2 cells); green LPCAT1 (late tip and AT2 cells). (E) Sections from control ( Nkx2.1-Cre; Stat3 +/fx ) and mutant ( Nkx2.1-Cre; Stat3 Δ/fx ) lungs with and without IL6. n =9 Nkx2.1-Cre; Stat3 Δ/fx lungs analysed in three independent experiments. Top panels: green, pro-SFTPC; red, LAMP3. Lower panels: red, pSTAT3; white, E-CAD (epithelium). Blue, DAPI. Dashed line, edge of lung. Scale bars: 50 μm in A,D; 100 μm in C,E.

Article Snippet: Primary antibodies: acetylated tubulin (mouse, 1:3000, Sigma, T7451), CEBPA (rabbit, 1:500, Santa Cruz, sc-61), cleaved caspase 3 (rabbit, 1:100, Abcam, ab2302), E-CAD (rat, 1:1000, Invitrogen, 13-1900; or mouse, 1:1000, BD Biosciences, 610182), GFP (chick, 1:1000, Abcam, AB13970), GR (rabbit, 1:100, Santa Cruz, sc-1004), HOPX (rabbit, 1:50, Santa Cruz, sc-30216, clone FL-73), KI67 (mouse, 1:200, BD, 550609), LAMP3 (rat, 1:100, Dendritics, DDX0192, clone 1006F7.05), LIF (goat, 1:100, R&D Systems, AB-449-NA), LPCAT1 (rabbit, 1:500, Proteintech), PDPN (hamster, 1:1000, DSHB, 8.1.1), RFP (rabbit, 1:250, Rockland, 600-401-379), pro-SFTPC (rabbit, 1:500, Millipore, AB3786), SOX2 (goat, 1:250, Santa Cruz, sc-17320, clone Y-17), SOX9 (goat, 1:200, R&D Systems, AF3075), pSTAT3-Tyr705 (rabbit, 1:200, Cell Signaling, 9145), STAT5a (rabbit, 1:20, Abcam, ab7968).

Techniques: Activation Assay, Control, Mutagenesis