Fig 2: AAV-PHP.B-Mediated Brain-wide TRIM9 Expression Ameliorates Ischemic Injury in C57BL/6J Middle-Aged Mice(A and B) Representative immunoblots (A) and quantification (B) of TRIM9 in brain tissue extracts of the ischemic brain hemispheres of C57BL/6J young or aged mice upon 30-min MCAO, followed by 12-hr reperfusion. Whole-tissue lysates were used for immunoblotting with antibodies against TRIM9 and actin. Isoform b was used for quantification. n = 4 mice per group. *p < 0.05 by one-way ANOVA and Bonferroni’s post hoc test. ns, non-significant.(C–F) Representative confocal images and quantifications of IL-6-positive (C and D) and CCL2-positive (E and F) neurons (NeuN) in the ischemic ipsilateral cortex regions of 12-week-old (12W) and 70-week-old (70W) C57BL/6J mice 12 hr after 30-min MCAO. Scale bar, 30 µm. n = 4 mice per group. **p < 0.01 by Student’s t test.(G) Representative cresyl violet staining images of the brain sections (at +1.6, +0.8, 0, -0.8, or -1.6 mm from the bregma) of recombinant AAV-infected aging mice that were tested 24 hr after 30-min MCAO. The area within the closed line indicates injured tissue. Scale bar, 30 µm. n = 8 mice per group.(H and I) Quantification of infarct volume (H) and motor neurological scores (I) of individual mice described in (G). The dashed line indicates the mean value from Trim9+/+ mice in Figure 2c. *p < 0.05 and **p < 0.01 by Mann-Whitney U test. ns, non-significant.(J and K) Representative confocal images (J) and quantification (K) of p-p65-positive neuronal cells in the peri-infarct regions of ischemic brain of recombinant AAV-infected aging mice 24 hr after 30-min MCAO. Scale bar, 30 µm. n = 8 mice per group. **p < 0.01 by Mann-Whitney U test.(L and M) Representative confocal images (L) and quantifications (M) of neuronal death based on a TUNEL assay in the infarct region of ischemic brain of aging mice injected with the indicated AAV 24 hr after 30-min MCAO. Scale bar, 30 mm. n = 5 mice per group. **p < 0.01 by Mann-Whitney U test.(N) Correlation between the p-p65-positive neuron population level and the motor neurological behavior score in PHP.B-GFP- or PHP.B-TRIM9-infected aged mice 24 hr after 30-min MCAO.Data in (B), (D), (F), (H), (I), (K), (M), and (N) are shown as mean ± SD.

Fig 3: TRIM9 Suppresses NF-?B Signaling by Competing with I?Ba for ß-TrCP Interaction(A and B) Representative immunoblots (A) and quantification (B) of I?Ba in primary cortical neurons isolated from Trim9+/+ and Trim9-/- embryos stimulated by 30 or 60 min of oxygen-glucose deprivation (OGD) followed by reoxygenation (R24h). Whole-cell lysates (WCLs) were used for immunoblotting with anti-TRIM9 and anti-actin antibody. **p < 0.01 by Student’s t test.(C and D) Representative images (C) and quantification (D) of Live-Dead viability assays 24 hr after 30-min OGD stimulation of human neural progenitor cell (hNPC)-derived neurons transduced with scramble or TRIM9-specific shRNA lentivirus. Scale bar, 30 µm.(E) Representative immunoblots of the ß-TrCP-TRIM9 interaction. Ischemic brain tissues of Trim9+/+ and Trim9-/- mice 24 hr after 30-min MCAO were used for immunoprecipitation (IP) with anti-ß-TrCP, followed by immunoblotting (IB) with anti-ß-TrCP or anti-TRIM9. WCLs were also used for IB with various antibodies. Ct Ig, isotype control immunoglobulin.(F and G) Representative immunoblot (F) and quantification (G) of I?Ba in Trim9-/- primary neurons transduced with lentivirus carrying vector (Vec), mouse hemagglutinin (HA)-TRIM9 wild-type (WT), or HA-TRIM9 SA mutant 24 hr after 30- or 60-min OGD. **p < 0.01 by Student’s t test. ns, non-significant.(H and I) Comparison between the ß-TrCP-TRIM9 interaction and the ß-TrCP-I?Ba interaction (H) and I?Ba ubiquitination (I). Trim9-/- primary neurons transduced with Vec, TRIM9 WT, or TRIM9 SA mutant lentivirus were stimulated with mock (-) or TNF-a (25 ng/ml) + MG312 (10 µM) (+) for 1 hr (see STAR Methods for details). WCLs were used for IP with anti-ß-TrCP, followed by IB with anti-I?Ba or anti-HA (H) or for IP with anti-I?Ba, followed by IB with anti-ubiquitin antibody (I). WCLs were also used for IB with antibodies against I?Ba, p-I?Ba, HA, ß-TrCP, TRIM9, or actin.(J) qRT-PCR analysis of a panel of inflammatory mediators, including IL-6 and CCL2, in Trim9+/+ (blue) and Trim9-/- (red) primary neurons primary neurons 24 hr after 30-min OGD treatment. **p < 0.01 and *p < 0.05 by one-way ANOVA and Bonferroni’s post hoc test.Data in (B), (D), (G), and (J) are from three independent experiments, shown as mean ± SD.See also Figures S4 and S5.

Fig 4: Exosomes derived from EGCG-treated tumor cells suppresses NF-?B pathway and inhibits M2 polarization of tumor-associated macrophages, which is dependent on up-regulated miR-16 in EGCG-treated tumor exosomes. (A) Tumor-associated macrophages were isolated from established murine breast cancer 4T1 cell lines, and treated with exosomes extracted from EGCG (100 µM)-treated and untreated 4T1 cells for 24 hours. Whole cell lysates were prepared and submitted to Western blot using anti-IKKa, anti-I-?B or anti-actin antibodies. (B) 4T1 cells were incubated with EGCG (100 µM) and simultaneously (24 hours) transfected with scramble or miR-16 inhibitor for 24 hours. Exosomes were then extracted from each tumor cells. Tumor-associated macrophages isolated from established murine breast cancer were treated with each exosome as indicated at the concentration of 50 µg/ml for 24 hours, and whole cell lysates were prepared and submitted to Western blot using anti-IKKa or anti-actin antibodies. The numbers in Figure 4A and 4B report the quantification of bands by imaging analysis with KODAK Molecular Imaging Software (MI). (C) 4T1 cells were incubated with EGCG (100 µM) and were simultaneously (24 hours) transfected with scramble or miR-16 inhibitor for 24 hours. Exosomes were then extracted from each tumor cells. Tumor-associated macrophages isolated from established murine breast cancer were treated with each exosome at the concentration 50 mg/ml for 24 hours, and total cellular RNA was extracted and submitted to RT-qPCR analysis for M2high cytokines (IL-6 and TGF-ß) and M2low cytokine (TNF-a). Histogram shows the relative expression of molecules compared to GAPDH as internal control. (D) Tumor cells were isolated and total RNA from cells were extracted and subjected to RT-qPCR to measure the miR-16 level. Histogram shows the relative expression of miR-16 compared to U6 as an internal control.

Fig 5: EGCG suppresses tumor growth, macrophages infiltration, and M2 polarization in in vivo murine breast cancer model. (A) Balb/c mice were challenged with 1× 105 murine breast cancer cell lines, 4T1, by subcutaneous injection into the right flanks. EGCG (10 mg/kg) or PBS (as a negative control) was administrated intraperitoneally (i.p.) on days 7, 9, and 11. Tumour sizes were measured every week until day 30 post tumour challenge. (B) Mice were sacrificed and the tumors were dissected and the weight was measured. (C) For evaluation of tumor-associated macrophages and M2 macrophage infiltration, immunohistochemical staining for CD68 and CD163 was done using tumor tissue. (D) Tumor cells were isolated and total RNA was extracted and then subjected to RT-qPCR to measure the levels of CSF-1, CCL-2, and GAPDH. Histogram shows the relative expression of molecules compared to GAPDH as internal control. (E) Tumor-associated macrophages were isolated, and total RNA from these cells were extracted and subjected to RT-qPCR to detect levels of M2high cytokines (IL-6 and TGF-ß) and M2low cytokine (TNF-a). Histogram shows the relative expression of molecules compared to GAPDH as internal control. (F) Tumor-associated macrophages were isolated and total RNA from cells were extracted and then subjected to RT-qPCR to detect the level of IKKa and GAPDH (internal control).