Journal: Heliyon

Article Title: Follicular fluid aids cell adhesion, spreading in an age independent manner and shows an age-dependent effect on DNA damage in fallopian tube epithelial cells

doi: 10.1016/j.heliyon.2024.e27336

Figure Lengend Snippet: Proteomic analysis of FF samples revealed extracellular matrix (ECM) protein - vitronectin as the component responsible for FTE adhesion and spreading A. Experimental workflow of proteomics experiment. B. Venn diagram showing 14 common proteins identified between young and aged FF samples from proteomics analysis. Common proteins are listed with vitronectin (highlighted in red). C. Representative immunoblot for vitronectin expression in 3 young (Y1–Y3) and 3 aged (A1-A3) FF samples. FF samples (5 μg) and recombinant vitronectin protein (0.01 μg, 0.1 μg, 1 μg) diluted with lysis buffer were used for immunoblotting. Arrow represents the band analyzed for vitronectin. Ponceau staining was used for loading control.

Article Snippet: Anti-rabbit vitronectin , Proteintech 15833-1-AP , 1:1000.

Techniques: Western Blot, Expressing, Recombinant, Lysis, Staining, Control

Journal: Heliyon

Article Title: Follicular fluid aids cell adhesion, spreading in an age independent manner and shows an age-dependent effect on DNA damage in fallopian tube epithelial cells

doi: 10.1016/j.heliyon.2024.e27336

Figure Lengend Snippet: Vitronectin in FF aids in FTE adhesion and spreading A. Representative brightfield images of FT190 cells seeded on ULA plates coated with FF sample (400 μl) and recombinant vitronectin protein (1 μg/well) for 4 h. Wells were washed with 1X PBS and FT190 cells were seeded on the coated plates. Images were acquired after 24 h. Scale bar = 200 μm. B. Cell proliferation was measured using an SRB assay to measure cell viability of FT190 cells on FF and vitronectin coated ULA plates. C. Representative Z stack images acquired by confocal microscopy of FT190 spheroids (labelled with Cell tracker 594) on NOF151 cells (labelled with Cell tracker 488) with FF samples and recombinant vitronectin protein (1 μg) (24 h). Scale bar = 200 μm. D. A maximum intensity projection re-construction of a three-dimensional FTE spheroid (labelled with Cell tracker 594) optical data stack over the surface of NOF151 cells (labelled with Cell tracker 488) treated with FF samples (Y1, A1) and vitronectin (1 μg). Spheroids were imaged with a 10× objective and re-constructed using the Imaris software. E. Representative images acquired by confocal microscopy showing a side and top projection of the FT190 spheroids on NOF151 cells with FF samples and recombinant vitronectin protein (1 μg) (24 h). Scale bar = 100 μm. F. Spheroid area (with and without FF/vitronectin) was determined by quantification of red fluorescent intensity using a polygon area measurement tool of the Imaris software.

Article Snippet: Anti-rabbit vitronectin , Proteintech 15833-1-AP , 1:1000.

Techniques: Recombinant, Sulforhodamine B Assay, Confocal Microscopy, Construct, Software

Journal: Heliyon

Article Title: Follicular fluid aids cell adhesion, spreading in an age independent manner and shows an age-dependent effect on DNA damage in fallopian tube epithelial cells

doi: 10.1016/j.heliyon.2024.e27336

Figure Lengend Snippet: Primary antibodies.

Article Snippet: Anti-rabbit vitronectin , Proteintech 15833-1-AP , 1:1000.

Techniques:

Journal: Iranian Journal of Microbiology

Article Title: Yeast-mediated display: probing Helicobacter pylori HopQ and CEACAM1 interaction

doi: 10.18502/ijm.v17i6.20366

Figure Lengend Snippet: SDS-PAGE and Western blot analyses. In panel A, the left image shows Coomassie Blue staining of S. cerevisiae expressing Aga2-C1ND (lanes 1 and 2). The middle blot shows two distinct bands at 50 kDa and 24 kDa detected using an HRP-conjugated anti-GFP antibody. Lane 1 corresponds to S. cerevisiae expressing the GFP-C1ND fusion construct. The presence of a 27 kDa band suggests cleavage of EGFP from its fusion partner. The right image shows signals detected by the anti-C1ND antibody for non-EGFP-tagged C1ND. The 24 kDa band corresponds to the Aga2-C1ND fusion protein. In panel B, the left image shows positive signals detected using the anti-C1ND antibody. Lane 1 shows the expression of the GFP-C1ND fusion protein, with bands at 24 kDa and 50 kDa. Lanes 2 and 3 represent strains expressing the Aga2-C1ND fusion protein, showing a band at 24 kDa. The positive control (lane 4) expresses the intact C1ND fragment, displaying a band at 12 kDa. The right image shows detection of the EGFP-C1ND fusion protein (50 kDa, lane 1) in a positive transformant using an HRP-conjugated anti-His-tag antibody. The positive control in this blot (lane 2) corresponds to an irrelevant His-tagged protein. In all blots, “C−“ indicates non-transformed lysate. Blots in panels A and B were developed using ECL and DAB staining, respectively.

Article Snippet: Immunoblots were probed overnight at 4°C with HRP-labeled antibodies against GFP (1:2000; Acris, USA), His-tag (1:1000; Sigma, USA), and C1ND (1:3000; Sigma, USA).

Techniques: SDS Page, Western Blot, Staining, Expressing, Construct, Positive Control, Transformation Assay

Journal: Iranian Journal of Microbiology

Article Title: Yeast-mediated display: probing Helicobacter pylori HopQ and CEACAM1 interaction

doi: 10.18502/ijm.v17i6.20366

Figure Lengend Snippet: Confocal microscopy images showing the surface expression of the Aga2-EGFP-G4S-C1ND fusion protein in S. cerevisiae , indicated by arrows. (A) Green fluorescence from the EGFP tag. (B) Red surface fluorescence of recombinant yeast cells stained with PE-conjugated anti-GFP antibody. (C) Merged image of panels A and B. (D) Brightfield image. (E) No fluorescence in the negative control. Scale bar: 5 μm

Article Snippet: Immunoblots were probed overnight at 4°C with HRP-labeled antibodies against GFP (1:2000; Acris, USA), His-tag (1:1000; Sigma, USA), and C1ND (1:3000; Sigma, USA).

Techniques: Confocal Microscopy, Expressing, Fluorescence, Recombinant, Staining, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Biological boundary conditions regulate the internalization of Aspergillus fumigatus conidia by alveolar cells

doi: 10.3389/fcimb.2025.1515779

Figure Lengend Snippet: Internalization of A fumigatus conidia in a subpopulation of Lamp + Actin + vesicles in cells on circular micropatterns and effect of ECM composition on conidia internalization. (A) Representative example of two cells on fibronectin-coated circular micropattern (28 µm). Overview of Lamp1 and Actin is shown as maximal Z-projection of FITC/TRITC channels of 9 focal planes. Magnified images show conidia in Lamp1 + (green) and Lamp1 + Actin + vesicles (red and green) 3 hours post-infection. Scale bars = 10 µm. (B) Percentage of conidia internalized in Lamp1 + Actin + vesicles relative to the total conidia number in cells constrained on fibronectin-coated circular micropatterns with different sizes and cell density at 1 and 3 hours post-infection. (C) Conidia spatial distribution map of 50 overlaid micropatterns displaying Lamp1 + Actin + vesicles with internalized conidia (green spheres in red circles). (D) Comparison of vitronectin- and fibronectin micropatterns. Quantification of the average number of total conidia and the percentage of conidia internalized in Lamp1 + and Lamp1 + Actin + vesicles at 1 and 3 hours post-infection relative to the total conidia number. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns P > 0.05. VN, vitronectin; FN, fibronectin. See also Supplementary Figure 1 .

Article Snippet: The patterned substrates were subsequently incubated with 150 μl of fibronectin (10 μg/ml, Sigma Aldrich, 341631, Austria) in PBS or vitronectin (3.3 μl/ml, ProSci, 91-362, USA) at 4°C overnight and washed once with PBS.

Techniques: Infection, Comparison

Journal: Nature Communications

Article Title: Serum/glucocorticoid-inducible kinase 1 deficiency induces NLRP3 inflammasome activation and autoinflammation of macrophages in a murine endolymphatic hydrops model

doi: 10.1038/s41467-023-36949-4

Figure Lengend Snippet: a Representative confocal microscopy images showing IBA1 (red) and DAPI (blue) staining in the ampulla and macula of AN ( n = 5) and MD patients ( n = 5). Analysis of images is shown on the right, quantifying the number of IBA1-positive cells per area. The right panels represent 2-fold enlargements of the white-boxed areas in the left panels. Scale bar = 25 μm. b Representative images of immunohistochemical staining and densitometric quantification of IL-1β, IL-18, and Casp-1 in the ampulla and macula of AN ( n = 4) and MD patients ( n = 4), scale bar = 100 μm. c Quantitative real-time PCR analysis of IL1B, IL18 , and CASP1 mRNA in VEO of AN ( n = 9) and MD patients ( n = 9). d ELISA of plasma IL-1β, IL-18, and Casp-1 levels in controls ( n = 20) and MD patients ( n = 20). e Quantitative real-time PCR analysis of SGK1 mRNA in VEO of AN ( n = 9) and MD patients ( n = 9). f Quantitative real-time PCR analysis of SGK1 mRNA in VRML cells of AN and MD patients ( n = 3). g Representative images of western blotting detecting NLRP3 and SGK1 in VRML cells of AN and MD patients ( n = 3). h Representative confocal microscopy images showing IL-1β (green), SGK1 (red), IBA1 (gray), and DAPI (blue) staining in the ampulla and macula of AN and MD patients, scale bar = 10 μm. i Immunofluorescence staining for Sox2 (green) and Myosin VIIa (red) in basilar membrane of neonatal mice in vitro, after treatment with IL-1β (10 ng/mL, 24 h). White asterisks indicate inner hair cell loss. Scale bar = 50 μm. j Immunofluorescence staining for TUNEL (green) and Myosin VIIa (red) in the basilar membrane at P3, after treatment with IL-1β (10 ng/mL, 24 h), scale bar = 50 μm. In each box plot, the central mark indicates the median, the bottom and top edge of the box indicate the interquartile range, and the whiskers represent the max and minimum data point. Results are presented as mean ± SD. Statistical analyses were carried out via two-sided t -test for a – f . VEO vestibular end organs, hVRMLs human vestibular-resident macrophage-like cells, Casp-1 caspase-1. Source data are provided as a Source Data file.

Article Snippet: To evaluate the effect of SGK1 inhibition, we treated the EH mice model with SGK1 inhibitor GSK650394 (10 mM, 40 μL/d/mouse, p.a., MCE) .

Techniques: Confocal Microscopy, Staining, Immunohistochemical staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Western Blot, Immunofluorescence, Membrane, In Vitro, TUNEL Assay

Journal: Nature Communications

Article Title: Serum/glucocorticoid-inducible kinase 1 deficiency induces NLRP3 inflammasome activation and autoinflammation of macrophages in a murine endolymphatic hydrops model

doi: 10.1038/s41467-023-36949-4

Figure Lengend Snippet: a – d Primary mVRML cells from sgk1 +/+ or sgk1 −/− mice upon stimulation with LPS (200 ng/mL, 3 h) alone or with ATP (5 mM, 30 min). a Quantitative real-time PCR of indicated genes ( n = 3 biologically independent experiments). b Cell lysates were subjected to western blotting. c ELISA showing Casp-1 and IL-1β secretion ( n = 3 biologically independent experiments). d Confocal microscopy of NLRP3 (green) and DAPI (blue) staining, scale bar = 50 μm. e – h Control and SGK1 siRNA-transfected THP-1 cells were stimulated with LPS (200 ng/mL, 3 h) alone or with ATP (5 mM, 30 min). e Quantitative real-time PCR of indicated genes ( n = 3 biologically independent experiments). f Cell lysates were subjected to western blotting. g ELISA showing Casp-1 and IL-1β secretion ( n = 3 biologically independent experiments). h Confocal microscopy of ASC (green) and DAPI (blue) staining, scale bar = 50 μm. i Dot plot showing flow cytometry analysis of control and SGK1 siRNA-transfected THP-1 cells with or without LPS and ATP in the presence or absence of z-VAD-FMK (20 μM, 3.5 h) or Ac-YVAD-cmk (40 μM, 3.5 h). j Western blotting with control and SGK1 siRNA-transfected THP-1 cells with or without LPS and ATP in the presence or absence of z-VAD-FMK (20 μM, 3.5 h) or Ac-YVAD-cmk (40 μM, 3.5 h). Results are presented as mean ± SD. Statistical analyses were carried out via two-sided t -test for a , c , e , g . mVRMLs, mouse vestibular-resident macrophage-like cells; Casp-1 caspase-1. Source data are provided as a Source Data file.

Article Snippet: To evaluate the effect of SGK1 inhibition, we treated the EH mice model with SGK1 inhibitor GSK650394 (10 mM, 40 μL/d/mouse, p.a., MCE) .

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Staining, Control, Transfection, Flow Cytometry

Journal: Nature Communications

Article Title: Serum/glucocorticoid-inducible kinase 1 deficiency induces NLRP3 inflammasome activation and autoinflammation of macrophages in a murine endolymphatic hydrops model

doi: 10.1038/s41467-023-36949-4

Figure Lengend Snippet: a Immunofluorescence staining for DAPI (blue), NLRP3 (green), and SGK1 (red) in THP-1 cells that were stimulated with LPS (200 ng/mL, 3 h) and ATP (5 mM, 30 min), scale bar = 50 μm. b Representative confocal images of HEK-293T cells transiently expressing GFP-NLRP3 (green), FLAG-SGK1 (red), MYC-ASC (magenta), and staining DAPI (blue), which were stimulated with LPS (200 ng/mL, 3 h) and ATP (5 mM, 30 min), scale bar = 50 μm. c Immunofluorescence staining for DAPI (blue), NLRP3 (green), CD68 (red), and SGK1 (magenta) in primary mVRML cells of WT mice which were stimulated with LPS (200 ng/mL, 3 h) and ATP (5 mM, 30 min), scale bar = 10 μm. d Immunofluorescence staining for DAPI (blue), NLRP3 (green), IBA1 (red), and SGK1(magenta) in macula of MD patients, scale bar = 10 μm. e Co-IP analysis of the interaction of NLRP3 and SGK1 from the total lysates of THP-1 cells which were stimulated with LPS (200 ng/mL, 3 h) and ATP (5 mM, 30 min). f Co-IP analysis of the interaction between GFP-NLRP3 and FLAG-SGK1 from the total lysates of HEK-293T cells which were stimulated with LPS (200 ng/mL, 3 h) and ATP (5 mM, 30 min). g GST pull-down assay with GST-fused NLRP3 and in vitro translated SGK1. h Co-IP analysis of the interaction of NLRP3 and SGK1 from the total lysates of THP-1 cells which were stimulated with saline, LPS (200 ng/mL, 3 h) or LPS + ATP (5 mM, 30 min). i Domain-mapping experiment showing details of the NLRP3-SGK1 interaction in plasmid-transfected HEK-293T cells. mVRMLs, mouse vestibular-resident macrophage-like cells; Casp-1 caspase-1. Source data are provided as a Source Data file.

Article Snippet: To evaluate the effect of SGK1 inhibition, we treated the EH mice model with SGK1 inhibitor GSK650394 (10 mM, 40 μL/d/mouse, p.a., MCE) .

Techniques: Immunofluorescence, Staining, Expressing, Co-Immunoprecipitation Assay, Pull Down Assay, In Vitro, Saline, Plasmid Preparation, Transfection

Journal: Nature Communications

Article Title: Serum/glucocorticoid-inducible kinase 1 deficiency induces NLRP3 inflammasome activation and autoinflammation of macrophages in a murine endolymphatic hydrops model

doi: 10.1038/s41467-023-36949-4

Figure Lengend Snippet: a Co-IP analysis of the serine phosphorylation of NLRP3 from the total lysates of primary mVRML cells of sgk1 +/+ or sgk1 −/− mice upon stimulation with LPS (200 ng/mL, 3 h) and ATP (5 mM, 30 min). b Co-IP analysis of the serine phosphorylation of NLRP3 from the total lysates of control and SGK1 siRNA-transfected THP-1 cells, which were stimulated with LPS (200 ng/mL, 3 h) and ATP (5 mM, 30 min). c In vitro kinase assay results showing the serine phosphorylation of NLRP3 after incubation with active SGK1-S422D or inactive SGK1-S422A via western blotting analysis. d In vitro kinase assay results showing the serine phosphorylation of wild-type NLRP3 or NLRP3-S5A after incubation with active SGK1-S422D via western blotting analysis. a – d , Protein expression was quantified by gray scanning, n = 3 biologically independent experiments. a , b NLRP3 served as a loading control for western blotting. c , d GST served as a loading control for western blotting. e SDD-AGE assay showing NLRP3 oligomerization in control or SGK1 siRNA-transfected THP-1 cells stimulated with saline, LPS (200 ng/mL, 3 h) or LPS + ATP (5 mM, 30 min). f Co-IP analysis of interaction between GFP-NLRP3 and MYC-ASC in the absence and presence of FLAG-SGK1 in HEK-293T cells stimulation with LPS (200 ng/mL, 3 h) and ATP (5 mM, 30 min). g Co-IP analysis of interaction between NLRP3 and ASC in control or SGK1 siRNA-transfected THP-1 cells, which were stimulated with LPS (200 ng/mL, 3 h) and ATP (5 mM, 30 min). Results are presented as mean ± SD. Statistical analyses were carried out via two-sided t -test for a – d . mVRMLs, mouse vestibular-resident macrophage-like cells; Casp-1 caspase-1. Source data are provided as a Source Data file.

Article Snippet: To evaluate the effect of SGK1 inhibition, we treated the EH mice model with SGK1 inhibitor GSK650394 (10 mM, 40 μL/d/mouse, p.a., MCE) .

Techniques: Co-Immunoprecipitation Assay, Phospho-proteomics, Control, Transfection, In Vitro, Kinase Assay, Incubation, Western Blot, Expressing, Saline

Journal: Nature Communications

Article Title: Serum/glucocorticoid-inducible kinase 1 deficiency induces NLRP3 inflammasome activation and autoinflammation of macrophages in a murine endolymphatic hydrops model

doi: 10.1038/s41467-023-36949-4

Figure Lengend Snippet: a – i sgk1 +/+ and sgk1 −/− mice were treated with LPS or saline. a Representative images of mid-modiolar cochlear sections, scale bar = 200 μm. b Measurements of IR-L in cochlear half-turns I–IV ( n = 5 mice per group). c Representative serial ABR wave recordings and thresholds in response to click sounds ( n = 5 mice per group). d ABR thresholds in response to pure tone bursts across all frequencies tested (4, 8, 12, 16, 24, and 32 kHz) ( n = 5 mice per group). * P indicates p -values for sgk1 +/+ +LPS versus sgk1 −/− +LPS; # P indicates p -values for sgk1 −/− +LPS versus sgk1 −/− +Saline; † P indicates p -values for sgk1 +/+ +LPS versus sgk1 +/+ +Saline. e Representative click-evoked VEMPs waves, P1-N1 peak amplitudes, and the P1 (white triangle) and N1 (black triangle) peak latencies of VEMPs at 100 dB nHL ( n = 6 mice per group). f Quantification of rotarod test ( n = 6 mice per group). g ELISA for plasma Casp-1 and IL-1β levels ( n = 3 mice per group). h Western blot analysis showing NLRP3, SGK1, pro Casp1, Casp-1 p20, pro IL-1β and cleaved IL-1β levels in inner ears ( n = 3 mice per group). i Immunofluorescence staining for TUNEL (green) and Myosin VIIa (red) in the basilar membrane, scale bar = 50 μm. Results are presented as mean ± SD. Statistical analyses were carried out via one-way ANOVA for b , c , e – g , and two-way ANOVA for d . EH endolymphatic hydrops, IR-L Increase ratios (IR) of the length of the Reissner’s membrane, ABR auditory brainstem response, VEMPs vestibular-evoked myogenic potentials, P1 positive peak, N1 negative peak, Casp-1 caspase-1. Source data are provided as a Source Data file.

Article Snippet: To evaluate the effect of SGK1 inhibition, we treated the EH mice model with SGK1 inhibitor GSK650394 (10 mM, 40 μL/d/mouse, p.a., MCE) .

Techniques: Saline, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Western Blot, Immunofluorescence, Staining, TUNEL Assay, Membrane

Journal: Nature Communications

Article Title: Serum/glucocorticoid-inducible kinase 1 deficiency induces NLRP3 inflammasome activation and autoinflammation of macrophages in a murine endolymphatic hydrops model

doi: 10.1038/s41467-023-36949-4

Figure Lengend Snippet: a – i The WT mice were untreated or pretreated with GSK650394 and challenged with LPS or saline. a Representative images of mid-modiolar cochlear sections, scale bar = 200 μm. b Measurements of IR-L in cochlear half-turns I–IV ( n = 5 mice per group). c Representative serial ABR wave recordings and thresholds in response to click sounds ( n = 5 mice per group). d ABR thresholds in response to pure tone burst across all frequencies tested (4, 8, 12, 16, 24, and 32 kHz) ( n = 5 mice per group). * P indicates p -values for GSK650394 + LPS versus LPS; # P indicates p -values for GSK650394 + LPS versus GSK650394; † P indicates p -values for LPS versus Control. e Representative click-evoked VEMPs waves, P1-N1 peak amplitudes, and the P1 (white triangle) and N1 (black triangle) peak latencies of VEMPs at 100 dB nHL ( n = 5 mice per group). f Quantification of rotarod test ( n = 5 mice per group). g ELISA for plasma Casp-1 and IL-1β levels ( n = 3 mice per group). h Western blot analysis showing NLRP3, SGK1, pro Casp1, Casp-1 p20, pro IL-1β and cleaved IL-1β levels in inner ears ( n = 3 mice per group). i Immunofluorescence staining for TUNEL (green) and Myosin VIIa (red) in the basilar membrane, scale bar = 50 μm. Results are presented as mean ± SD. Statistical analyses were carried out via one-way ANOVA for b , c , e – g , and two-way ANOVA for d . EH endolymphatic hydrops, IR-L Increase ratios (IR) of the length of the Reissner’s membrane, ABR auditory brainstem response, VEMPs vestibular-evoked myogenic potentials, P1 positive peak, N1 negative peak, Casp-1 caspase-1. Source data are provided as a Source Data file.

Article Snippet: To evaluate the effect of SGK1 inhibition, we treated the EH mice model with SGK1 inhibitor GSK650394 (10 mM, 40 μL/d/mouse, p.a., MCE) .

Techniques: Saline, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Western Blot, Immunofluorescence, Staining, TUNEL Assay, Membrane

Journal: Nature Communications

Article Title: Serum/glucocorticoid-inducible kinase 1 deficiency induces NLRP3 inflammasome activation and autoinflammation of macrophages in a murine endolymphatic hydrops model

doi: 10.1038/s41467-023-36949-4

Figure Lengend Snippet: a – i The sgk1 +/+ and sgk1 −/− mice were treated with saline or MCC950, and followed by LPS stimulation. a Representative images of EH severity in mid-modiolar cochlear sections, scale bar = 200 μm. b Measurements of IR-L in cochlear half-turns I–IV ( n = 5 mice per group). c Representative serial ABR wave recordings and thresholds in response to click sounds ( n = 5 mice per group). d ABR thresholds in response to pure tone burst across all frequencies tested (4, 8, 12, 16, 24, and 32 kHz) ( n = 5 mice per group). * P indicates p -values for sgk1 −/− +LPS + MCC950 versus sgk1 −/− +LPS; # P indicates p -values for sgk1 +/+ +LPS versus sgk1 −/− +LPS; † P indicates p -values for sgk1 +/+ +LPS versus sgk1 −/− +LPS + MCC950. e Representative click-evoked VEMP waves, P1-N1 peak amplitudes, and the P1 (white triangle) and N1 (black triangle) peak latencies of VEMPs at 100 dB nHL ( n = 6 mice per group). f Quantification of rotarod test ( n = 6 mice per group). g Concentrations of Casp-1 and IL-1β in plasma of treated mice as measured via ELISA ( n = 3 mice per group). h Western blot analysis showing NLRP3, SGK1, pro Casp1, Casp-1 p20, pro IL-1β, and cleaved IL-1β levels in the ears ( n = 3 mice per group). i Immunofluorescence staining for TUNEL (green) and Myosin VIIa (red) in the basilar membrane, scale bar = 50 μm. Results are presented as mean ± SD. Statistical analyses were carried out via one-way ANOVA for b , c , e – g , and two-way ANOVA for d . EH endolymphatic hydrops, IR-L Increase ratios (IR) of the length of the Reissner’s membrane, ABR auditory brainstem response, VEMPs vestibular-evoked myogenic potentials, P1 positive peak, N1 negative peak, Casp-1 caspase-1. Source data are provided as a Source Data file.

Article Snippet: To evaluate the effect of SGK1 inhibition, we treated the EH mice model with SGK1 inhibitor GSK650394 (10 mM, 40 μL/d/mouse, p.a., MCE) .

Techniques: Saline, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Staining, TUNEL Assay, Membrane

Journal: Nature Communications

Article Title: Serum/glucocorticoid-inducible kinase 1 deficiency induces NLRP3 inflammasome activation and autoinflammation of macrophages in a murine endolymphatic hydrops model

doi: 10.1038/s41467-023-36949-4

Figure Lengend Snippet: Under normal conditions, SGK1 binds to the PYD domain of NLRP3 and phosphorylates NLRP3 at S5, thereby preventing the assembly of the mature inflammasome. SGK1 deficiency then augments NLRP3 inflammasome activation, which recruits ASC and pro caspase-1 to assemble the mature inflammasome, resulting in the maturation and secretion of IL-1β and IL-18 and induction of pyroptosis, thereby facilitating the severity of LPS-induced EH.

Article Snippet: To evaluate the effect of SGK1 inhibition, we treated the EH mice model with SGK1 inhibitor GSK650394 (10 mM, 40 μL/d/mouse, p.a., MCE) .

Techniques: Activation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ERK/GATAD2A/SAA1 axis drives osimertinib resistance via NF-κB and macrophage reprogramming in EGFR-mutated lung adenocarcinoma

doi: 10.1186/s13046-026-03792-6

Figure Lengend Snippet: Osimertinib upregulates SAA1, which associates with poor clinical outcomes in LUAD. A Schematic of short-term osimertinib exposure (IC 50 , 48 h) in three EGFR-mutant LUAD cell lines (PC-9, HCC827, H1975). Venn diagram illustrates consistently dysregulated secreted-protein genes across the three cell lines. B RNA-seq analysis identifying six differentially expressed secreted-protein genes, with FPKM shown in each dot. C - D Western blot, and ELISA assay validation of SAA1 upregulation, demonstrating a dose-dependent increase in PC-9 and H1975 cells ( n = 3). E Relative cell viability assays confirming ≥ 20-fold resistance to osimertinib in established OR cell lines (PC-9 OR , H1975 OR ) ( n = 3). F - G qPCR and western blot results of SAA1 expression across DTPCs and long-term OR models ( n = 3). H Representative IHC staining of SAA1 in normal lung tissue, LUAD tumors before TKI treatment, and TKI-treated LUAD samples. Scale bar, 20 μm. I Quantification of SAA1 IHC H-scores in paired EGFR-mutant LUAD samples collected before and after neoadjuvant osimertinib treatment ( n = 5). J Western blot analysis of paired EGFR-mutant LUAD tumors before (TKI-naïve) and after osimertinib (TKI-residual) treatment ( n = 5). K Kaplan-Meier analysis of disease-free survival of adjuvant osimertinib-treated LUAD patients ( n = 40). Dunnett’s multiple comparison tests were applied for panels D and F ; two-way ANOVA test for panel E; Paired Student’s t-test for panel I; and log-rank test for panel K. Abbreviations: PR, parental; OR, osimertinib-resistant; DTPC, drug-tolerant persister cells; TKI, tyrosine kinase inhibitor; IHC, immunohistochemistry

Article Snippet: Antibodies included: SAA1 (1:1,000; Proteintech, 16721-1-AP), β-Actin (1:2,500; Beyotime, AF0003), GAPDH (1:2,500; Servicebio, ZB15004-HRP), NF-κB (1:1,000; CST, 8242), p-NF-κB (Ser536) (1:1,000; CST, 3033), IκBα (1:1,000; CST, 4814), p-IκBα (Ser32/36) (1:1,000; CST, 9246), GATAD2A (1:2,000; Proteintech, 12294-1-AP), MTA2 (1:2,000; Proteintech, 17554-1-AP), MBD2 (1:2,000; Proteintech, 55200-1-AP), and p-GATAD2A (Ser114) (1:2,000; ABclonal, custom), TLR2 (1:2,000; Proteintech, 17236-1-AP), TLR4 (1:2,000; Proteintech, 19811-1-AP), anti-histone H3 (1:500; Servicebio, GB11102), RXRA (1:2,000; Proteintech, 21218-1-AP), YY1 (1:2,000; Proteintech, 66281-1-AP), ETV6 (1:2,000; Proteintech, 13182-1-AP), and MAX (1:2,000; Proteintech, 10426-1-AP).

Techniques: Mutagenesis, RNA Sequencing, Analysis, Western Blot, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Expressing, Immunohistochemistry, Tissue, Adjuvant, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ERK/GATAD2A/SAA1 axis drives osimertinib resistance via NF-κB and macrophage reprogramming in EGFR-mutated lung adenocarcinoma

doi: 10.1186/s13046-026-03792-6

Figure Lengend Snippet: SAA1 is required for both drug tolerance and established osimertinib resistance. A - B Relative cell viability and proliferation assays with different generations of EGFR-TKI (10 nM) in parental EGFR-mutant LUAD cell lines transfected with two independent siRNAs targeting SAA1 in PC-9 and H1975 cells ( n = 3). C Western blot confirming the establishment of SAA1-OE and SAA1-KO OR cell models generated by lentiviral transduction. D - E Relative cell viability and proliferation assays (10 nM) with different generations of EGFR-TKI in SAA1-OE, SAA1-KO OR models, and SAA1-KO cells rescued with exogenous SAA1 (100 ng/ml) ( n = 3). F Quantification of DTPC formation efficiency in LUAD cells treated with PBS or exogenous SAA1 (100 ng/ml) ( n = 3). G - H In vivo assessment using a PC-9 OR CDX model established with cells transduced with either a negative control vector or SAA1-KO lentivirus. Mice received daily oral gavage of osimertinib (5 mg/kg) or saline for 9 consecutive days ( n = 6). Tumor growth curves ( G ) and representative final tumor specimens ( H ) are shown. Dunnett’s multiple comparison tests were used for panels B and E , and two-way ANOVA test was applied for panel G . Abbreviations: SAA1-OE, SAA1 overexpression; SAA1-KO, SAA1 knockout; OR, osimertinib-resistant; Osi, osimertinib; Gef, gefitinib; Afa, afatinib; TKI, tyrosine kinase inhibitor; DTPC, drug-tolerant persister cells

Article Snippet: Antibodies included: SAA1 (1:1,000; Proteintech, 16721-1-AP), β-Actin (1:2,500; Beyotime, AF0003), GAPDH (1:2,500; Servicebio, ZB15004-HRP), NF-κB (1:1,000; CST, 8242), p-NF-κB (Ser536) (1:1,000; CST, 3033), IκBα (1:1,000; CST, 4814), p-IκBα (Ser32/36) (1:1,000; CST, 9246), GATAD2A (1:2,000; Proteintech, 12294-1-AP), MTA2 (1:2,000; Proteintech, 17554-1-AP), MBD2 (1:2,000; Proteintech, 55200-1-AP), and p-GATAD2A (Ser114) (1:2,000; ABclonal, custom), TLR2 (1:2,000; Proteintech, 17236-1-AP), TLR4 (1:2,000; Proteintech, 19811-1-AP), anti-histone H3 (1:500; Servicebio, GB11102), RXRA (1:2,000; Proteintech, 21218-1-AP), YY1 (1:2,000; Proteintech, 66281-1-AP), ETV6 (1:2,000; Proteintech, 13182-1-AP), and MAX (1:2,000; Proteintech, 10426-1-AP).

Techniques: Mutagenesis, Transfection, Western Blot, Generated, Transduction, In Vivo, Negative Control, Plasmid Preparation, Saline, Comparison, Over Expression, Knock-Out

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ERK/GATAD2A/SAA1 axis drives osimertinib resistance via NF-κB and macrophage reprogramming in EGFR-mutated lung adenocarcinoma

doi: 10.1186/s13046-026-03792-6

Figure Lengend Snippet: SAA1 activates the TLR4/NF-κB pathway to drive osimertinib resistance. A Heatmap showing the expression patterns of NF-κB-associated genes in PC-9 cells treated with exogenous SAA1 (100 ng/ml, 24 h) compared with PBS. B GSEA plot indicating enrichment of the NF-κB signaling pathway in SAA1-treated PC-9 cells. C - D Western blot analysis of whole-cell lysates ( C , assessing p65, p-p65, IκBα, and p-IκBα) and subcellular fractions ( D , assessing nuclear and cytoplasmic p65) in PC-9 OR and H1975 OR cells transfected with vector control, SAA1 overexpression, SAA1 knockout, or SAA1 knockout rescued with exogenous SAA1. E - F Western blot evaluating the effect of the NF-κB inhibitor BAY 11-7082 (5 µM) on SAA1-induced pathway activation in whole-cell lysates ( E ) and subcellular fractions ( F ). G - H Western blot evaluating the effect of the TLR4 inhibitor TAK-242 (10 µM) on SAA1-induced pathway activation in whole-cell lysates ( G ) and subcellular fractions ( H ). I - L Cell proliferation curves ( I , K ) and relative cell viability assays determining osimertinib IC50 ( J , L ) in PC-9 OR and H1975 OR cells treated with PBS or exogenous SAA1, in the presence or absence of BAY 11-7082 (5 µM) or TAK-242 (10 µM) ( n = 3). Two-way ANOVA tests were applied for panels I - L . Abbreviations: SAA1-OE, SAA1 overexpression; SAA1-KO, SAA1 knockout; OR, osimertinib-resistant; NF-κBi, NF-κB inhibitor; TLR4i, TLR4 inhibitor

Article Snippet: Antibodies included: SAA1 (1:1,000; Proteintech, 16721-1-AP), β-Actin (1:2,500; Beyotime, AF0003), GAPDH (1:2,500; Servicebio, ZB15004-HRP), NF-κB (1:1,000; CST, 8242), p-NF-κB (Ser536) (1:1,000; CST, 3033), IκBα (1:1,000; CST, 4814), p-IκBα (Ser32/36) (1:1,000; CST, 9246), GATAD2A (1:2,000; Proteintech, 12294-1-AP), MTA2 (1:2,000; Proteintech, 17554-1-AP), MBD2 (1:2,000; Proteintech, 55200-1-AP), and p-GATAD2A (Ser114) (1:2,000; ABclonal, custom), TLR2 (1:2,000; Proteintech, 17236-1-AP), TLR4 (1:2,000; Proteintech, 19811-1-AP), anti-histone H3 (1:500; Servicebio, GB11102), RXRA (1:2,000; Proteintech, 21218-1-AP), YY1 (1:2,000; Proteintech, 66281-1-AP), ETV6 (1:2,000; Proteintech, 13182-1-AP), and MAX (1:2,000; Proteintech, 10426-1-AP).

Techniques: Expressing, Western Blot, Analysis, Transfection, Plasmid Preparation, Control, Over Expression, Knock-Out, Activation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ERK/GATAD2A/SAA1 axis drives osimertinib resistance via NF-κB and macrophage reprogramming in EGFR-mutated lung adenocarcinoma

doi: 10.1186/s13046-026-03792-6

Figure Lengend Snippet: SAA1 reshapes the tumor microenvironment by promoting macrophage recruitment and pro-tumorigenic polarization. A - B Representative immunofluorescence images ( A ) and corresponding quantification ( B ) of F4/80⁺ (green) and CD206⁺ (yellow) macrophages in tumors from PC-9 OR CDX models treated with vehicle or osimertinib, comparing vector control and SAA1-KO groups. Scale bar, 20 μm. C - D Flow cytometry plots ( D ) and statistical quantification ( C ) of M2 macrophage polarization (CD206⁺) in BMDM and THP-1 cells treated with PBS or exogenous SAA1 (100 ng/ml, 24 h). E qPCR analysis of classical M2 macrophage markers ( CD206 , VEGFA , TGFB1 , IL-10 , ARG1 ) in THP-1 and BMDM cells co-cultured with PC-9 OR cell lines under osimertinib treatment, comparing vector control and SAA1 knockout conditions ( n = 3). F qPCR analysis of newly proposed macrophage markers ( SPP1 , C1QC , ISG15 , FN1 ) in THP-1 and BMDM cells after exposure to exogenous SAA1(100 ng/ml, 24 h) ( n = 3). G qPCR analysis of M2 markers and SPP1 in THP-1 and BMDM cells treated with TAK-242 (10 µM, 24 h), with or without exogenous SAA1 (100 ng/ml, 24 h) ( n = 3). H - I Flow cytometry analysis ( H ) and comprehensive quantification ( I ) of CD206⁺ macrophages in THP-1 cells treated with PBS or exogenous SAA1 (100 ng/ml, 24 h), combined with DMSO, TAK-242 (10 µM, 24 h), or C29 (50 µM, 24 h), demonstrating the comparative blockade efficacy of distinct TLR pathways ( n = 3). J - L Representative multiplex immunofluorescence images ( L ) and quantitative analysis ( J , K ) of CD68⁺ (red), CD206⁺ (green), and SPP1⁺ (yellow) macrophage infiltration in paired LUAD patient samples before (TKI-naïve) and after (TKI-residual) osimertinib therapy. Scale bar, 50 μm. Student’s t-tests were for panels C , F , J , K and one-way or two-way ANOVA with Dunnett’s multiple comparison tests were for panels B, E, G, I. Abbreviations: OR, osimertinib-resistant; SAA1-KO, SAA1 knockout; Exo, exogenous; TKI, tyrosine kinase inhibitor; IHC, immunohistochemistry

Article Snippet: Antibodies included: SAA1 (1:1,000; Proteintech, 16721-1-AP), β-Actin (1:2,500; Beyotime, AF0003), GAPDH (1:2,500; Servicebio, ZB15004-HRP), NF-κB (1:1,000; CST, 8242), p-NF-κB (Ser536) (1:1,000; CST, 3033), IκBα (1:1,000; CST, 4814), p-IκBα (Ser32/36) (1:1,000; CST, 9246), GATAD2A (1:2,000; Proteintech, 12294-1-AP), MTA2 (1:2,000; Proteintech, 17554-1-AP), MBD2 (1:2,000; Proteintech, 55200-1-AP), and p-GATAD2A (Ser114) (1:2,000; ABclonal, custom), TLR2 (1:2,000; Proteintech, 17236-1-AP), TLR4 (1:2,000; Proteintech, 19811-1-AP), anti-histone H3 (1:500; Servicebio, GB11102), RXRA (1:2,000; Proteintech, 21218-1-AP), YY1 (1:2,000; Proteintech, 66281-1-AP), ETV6 (1:2,000; Proteintech, 13182-1-AP), and MAX (1:2,000; Proteintech, 10426-1-AP).

Techniques: Immunofluorescence, Plasmid Preparation, Control, Flow Cytometry, Analysis, Cell Culture, Knock-Out, Multiplex Assay, Comparison, Immunohistochemistry, Co-Culture

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ERK/GATAD2A/SAA1 axis drives osimertinib resistance via NF-κB and macrophage reprogramming in EGFR-mutated lung adenocarcinoma

doi: 10.1186/s13046-026-03792-6

Figure Lengend Snippet: GATAD2A S114 phosphorylation modulates NuRD complex integrity and regulates SAA1 expression. A DNA pull-down assay using a biotin-labeled SAA1 promoter probe (-1000 to + 200 bp) incubated with PC-9 nuclear extracts. Mass spectrometry identified 41 transcription factors, five of which (RXRA, YY1, ETV6, MAX, GATAD2A) were selected based on ENCODE ChIP-seq data. B qPCR of SAA1 expression following two individual siRNA knockdown of the five candidate transcription factors in PC-9 cells ( n = 3). C , D qPCR and western blot of SAA1 levels in PC-9 and H1975 cells transfected with two independent siRNAs targeting GATAD2A ( n = 3). E Predicted GATAD2A binding motif and binding peaks in the SAA1 promoter region from public ChIP-seq datasets, with indicated the location of the primer used for ChIP-qPCR validation. F ChIP-qPCR of GATAD2A occupancy at the SAA1 promoter in PC-9 and H1975 cells compared with IgG controls ( n = 3). G Luciferase reporter assay of wild-type and mutant SAA1 promoters in PC-9 and H1975 cells following GATAD2A knockdown ( n = 3). H Phosphoproteomic mapping of GATAD2A in PC-9 parental and OR cells, showing phosphorylated residues including S107, S110 and S114, among which S114 was predicted to be phosphorylated by ERK. I SAA1 expression in PC-9 and H1975 cells harboring the GATAD2A S114A knock-in compared with wild-type cells following osimertinib treatment (10 nM, 48 h). J Co-immunoprecipitation analysis of GATAD2A interactions with MBD2 and MTA2 in PC-9 and H1975 cells under different treatments, including osimertinib (10 nM, 48 h), SCH772984 (ERK inhibitor) (10 µM, 48 h), and S114A mutation. K Quantitative analysis of the PLA signals representing GATAD2A-NuRD complex interactions in PC-9 and H1975 cells under the indicated conditions ( n = 3). L PLA showing GATAD2A-NuRD component interactions under the same conditions as panel J . Dunnett’s multiple comparison tests were applied for panels B , C , G , and K ; Student’s t-test for panel F . Abbreviations: TF, transcription factors; ChIP, chromatin immunoprecipitation; PLA, proximity ligation assay; NuRD, nucleosome remodeling and deacetylase complex

Article Snippet: Antibodies included: SAA1 (1:1,000; Proteintech, 16721-1-AP), β-Actin (1:2,500; Beyotime, AF0003), GAPDH (1:2,500; Servicebio, ZB15004-HRP), NF-κB (1:1,000; CST, 8242), p-NF-κB (Ser536) (1:1,000; CST, 3033), IκBα (1:1,000; CST, 4814), p-IκBα (Ser32/36) (1:1,000; CST, 9246), GATAD2A (1:2,000; Proteintech, 12294-1-AP), MTA2 (1:2,000; Proteintech, 17554-1-AP), MBD2 (1:2,000; Proteintech, 55200-1-AP), and p-GATAD2A (Ser114) (1:2,000; ABclonal, custom), TLR2 (1:2,000; Proteintech, 17236-1-AP), TLR4 (1:2,000; Proteintech, 19811-1-AP), anti-histone H3 (1:500; Servicebio, GB11102), RXRA (1:2,000; Proteintech, 21218-1-AP), YY1 (1:2,000; Proteintech, 66281-1-AP), ETV6 (1:2,000; Proteintech, 13182-1-AP), and MAX (1:2,000; Proteintech, 10426-1-AP).

Techniques: Phospho-proteomics, Expressing, Pull Down Assay, Labeling, Incubation, Mass Spectrometry, ChIP-sequencing, Knockdown, Western Blot, Transfection, Binding Assay, ChIP-qPCR, Biomarker Discovery, Luciferase, Reporter Assay, Mutagenesis, Knock-In, Co-Immunoprecipitation Assay, Analysis, Comparison, Chromatin Immunoprecipitation, Proximity Ligation Assay, Histone Deacetylase Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ERK/GATAD2A/SAA1 axis drives osimertinib resistance via NF-κB and macrophage reprogramming in EGFR-mutated lung adenocarcinoma

doi: 10.1186/s13046-026-03792-6

Figure Lengend Snippet: Preclinical evaluation of Anti-SAA1 neutralization in EGFR-mutant models. A Proliferation curves of PC-9 OR and H1975 OR cells treated with Anti-SAA1 (1 µg/mL) or isotype IgG control across the indicated time points ( n = 3). B Relative cell viability of PC-9 OR and H1975 OR cells following Anti-SAA1 (1 µg/mL) or IgG treatment ( n = 3). C - D Representative tumor images ( C ) and tumor growth curves ( D ) of PC-9 OR -derived CDX models treated with control therapy, Anti-SAA1, osimertinib, or the combination ( n = 6). Mice were administered daily oral gavage of osimertinib (5 mg/kg) or saline for nine consecutive days. In parallel, intraperitoneal injections of Anti-SAA1 (0.25 µg/g) or control IgG were given every three days ( n = 6). E Morphological features of PDOs treated with IgG, Anti-SAA1 (1 µg/mL) combined with either saline or osimertinib (10 nM). Scale bar, 20 μm. F ATP levels in corresponding PDOs under the indicated treatment conditions ( n = 3). G Representative tumor images of PDX models treated with control therapy, Anti-SAA1, osimertinib, or the combination ( n = 3 for each PDX). H Schematic of tumor induction in the h EGFR L858R/T790M genetically engineered mouse model using intratracheal AAV- Scgb1a1 -Cre delivery. I Micro-CT-based quantification of tumor burden after 4 weeks since treatment with osimertinib alone, Anti-SAA1 plus osimertinib, or isotype control. 30-days after AAV delivery, mice were administered osimertinib (5 mg/kg) or saline by daily oral gavage for 21 consecutive days. In addition, intraperitoneal injections of Anti-SAA1 (0.25 µg/g) or control IgG were given every three days, for a total of six doses. J Survival curves of mice receiving the indicated treatments ( n = 6). K Representative immunohistochemical and multiplex immunofluorescence images of lung tumor sections derived from immunocompetent GEMM models following the indicated treatments. DAPI (blue), F4/80 (magenta), CD206 (green), SPP1 (red), and CD8 (yellow). Scale bars, 50 μm. L - M Quantitative analyses of the percentage of immunosuppressive SPP1 + CD206 + subpopulations among total F4/80 + macrophages ( L ) and the infiltration rate of CD8 + T cells among total DAPI + cells ( M ) in the spontaneous tumor tissues ( n = 6). Two-way ANOVA for panels A , B , and D ; Dunnett’s multiple comparison test was applied for panel F , log-rank test for panel J , and one-way ANOVA for panels L and M . Abbreviations: Anti-SAA1, SAA1 neutralizing antibody; OR, osimertinib-resistant; CDX, cell-derived xenograft; PDO, patient-derived organoid; PDX, patient-derived xenograft; AAV, adeno-associated virus; micro-CT, micro-computed tomography

Article Snippet: Antibodies included: SAA1 (1:1,000; Proteintech, 16721-1-AP), β-Actin (1:2,500; Beyotime, AF0003), GAPDH (1:2,500; Servicebio, ZB15004-HRP), NF-κB (1:1,000; CST, 8242), p-NF-κB (Ser536) (1:1,000; CST, 3033), IκBα (1:1,000; CST, 4814), p-IκBα (Ser32/36) (1:1,000; CST, 9246), GATAD2A (1:2,000; Proteintech, 12294-1-AP), MTA2 (1:2,000; Proteintech, 17554-1-AP), MBD2 (1:2,000; Proteintech, 55200-1-AP), and p-GATAD2A (Ser114) (1:2,000; ABclonal, custom), TLR2 (1:2,000; Proteintech, 17236-1-AP), TLR4 (1:2,000; Proteintech, 19811-1-AP), anti-histone H3 (1:500; Servicebio, GB11102), RXRA (1:2,000; Proteintech, 21218-1-AP), YY1 (1:2,000; Proteintech, 66281-1-AP), ETV6 (1:2,000; Proteintech, 13182-1-AP), and MAX (1:2,000; Proteintech, 10426-1-AP).

Techniques: Neutralization, Mutagenesis, Control, Derivative Assay, Saline, Micro-CT, Immunohistochemical staining, Multiplex Assay, Immunofluorescence, Comparison, Xenograft Assay, Virus

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ERK/GATAD2A/SAA1 axis drives osimertinib resistance via NF-κB and macrophage reprogramming in EGFR-mutated lung adenocarcinoma

doi: 10.1186/s13046-026-03792-6

Figure Lengend Snippet: Schematic representation illustrating the mechanism of the ERK/GATAD2A/SAA1 axis in driving osimertinib resistance in EGFR-mutated lung adenocarcinoma. The targeted inhibition of mutant EGFR by osimertinib suppresses downstream ERK signaling. This reduction in ERK kinase activity leads to the dephosphorylation of the epigenetic regulator GATAD2A at the S114 site. In its dephosphorylated state, GATAD2A fails to maintain the assembly of the NuRD corepressor complex at the SAA1 promoter. The dissociation of the NuRD complex relieves the epigenetic silencing of the SAA1 gene, resulting in robust transcriptional derepression and the subsequent secretion of SAA1 protein into the extracellular space. Once secreted, SAA1 functions as a dual-modal mediator to orchestrate acquired resistance. Autocrinely, SAA1 binds to TLR4 on the tumor cell membrane, triggering the intracellular NF-κB signaling cascade to promote tumor cell survival and persistence. Paracrinely, SAA1 remodels the tumor microenvironment by promoting the polarization of macrophages toward an immunosuppressive pro-tumor phenotype. Therapeutic intervention with an anti-SAA1 neutralizing antibody effectively intercepts these extracellular signals, abrogating both the tumor-intrinsic NF-κB survival pathway and the macrophage-mediated immune evasion, thereby restoring sensitivity to osimertinib

Article Snippet: Antibodies included: SAA1 (1:1,000; Proteintech, 16721-1-AP), β-Actin (1:2,500; Beyotime, AF0003), GAPDH (1:2,500; Servicebio, ZB15004-HRP), NF-κB (1:1,000; CST, 8242), p-NF-κB (Ser536) (1:1,000; CST, 3033), IκBα (1:1,000; CST, 4814), p-IκBα (Ser32/36) (1:1,000; CST, 9246), GATAD2A (1:2,000; Proteintech, 12294-1-AP), MTA2 (1:2,000; Proteintech, 17554-1-AP), MBD2 (1:2,000; Proteintech, 55200-1-AP), and p-GATAD2A (Ser114) (1:2,000; ABclonal, custom), TLR2 (1:2,000; Proteintech, 17236-1-AP), TLR4 (1:2,000; Proteintech, 19811-1-AP), anti-histone H3 (1:500; Servicebio, GB11102), RXRA (1:2,000; Proteintech, 21218-1-AP), YY1 (1:2,000; Proteintech, 66281-1-AP), ETV6 (1:2,000; Proteintech, 13182-1-AP), and MAX (1:2,000; Proteintech, 10426-1-AP).

Techniques: Inhibition, Mutagenesis, Activity Assay, De-Phosphorylation Assay, Membrane, Sensitivity