Fig 1: RASSF1A interacts with HDAC6 to enhance acetylation of α-Tubulin and co-localizes with acetylated microtubules. a, b Representative immunoblot (a) and quantification (b) showing levels of acetylated α-tubulin (Ac-α-Tub) in liver tissues from 4-month-old wild-type and RASSF1A−/− mice. c, d Representative immunoblot (c) and quantification (d) showing levels of acetylated α-tubulin in hepatocytes isolated from wild-type and RASSF1A−/− mice. e, f Representative immunoblot (e) and quantification (f) showing levels of acetylated α-tubulin in HeLa cells transiently transfected with control plasmid, plasmid expressing HA-RASSF1A (HA-RA), and plasmid expressing HA-RASSF1A∆ (HA-RA∆). g, h Representative images (g) and quantification (h) showing the immunostaining intensities of acetylated α-tubulin in cells similar to those in e. Bar = 20 µm. Red, HA-RA or HA-RA∆; green, acetylated a-tubulin; and yellow, colocalization. i, j Representative immunoblot (i) and quantification (j) showing the impact of RASSF1A on levels of HDAC6 in cells similar to those in e. k, l Representative immunoblots showing levels of Flag-HDAC6 coimmunoprecipitated with endogenous RASSF1A from HeLa cells (k) or exogenous RASSF1A in 293T cells (l) transiently overexpressing Flag-HDAC6. m Representative immunoblots showing levels of RA-HA or RA-RA∆ coimmunoprecipitated with Flag-HDAC6 in 293T cells. n Representative immunoblot showing levels of GFP fused fragments of RASSF1A coimmunoprecipitated with HDAC6 from lysates of HeLa cells transiently expressing GFP fused RASSF1A constructs
Fig 2: RASSF1A regulates autophagy initiation not through MAP1S. a A diagram showing the domain structures of RASSF1A protein and its mutant constructs. RASSF1A has four characterized domains: C1, phorbol ester/diacylglycerol binding domain; ATM, ataxia-telangiectasia mutated domain; RA*, Ras-association (RalGDS/AF-6) domain (F3); SARAH, MST and SAV1 binding domain (F4). Four fragments of RASSF1A (F1–4) were fused with GFP, respectively. RASSF1A with RA domain deleted (RAΔ) was fused with HA. b A diagram showing the domain structures of MAP1S protein and its mutant constructs. FL full length, HC heavy chain, SC short chain, LC light chain, 4G1 region recognized by MAP1S monoclonal antibody 4G1, HBD HDAC4‐binding domain (R653‐Q855), FLΔ full length MAP1S with HBD domain deleted. c, d Representative immunoblots (c) and quantification (d) showing the impact of RASSF1A on levels of MAP1S, HDAC4, Bcl-2 and P27 in liver tissues from 3 pairs of wild-type and RASSF1A−/− littermates. e, f Representative immunoblots (e) and quantification (f) showing the impact of RASSF1A on levels of acetylated MAP1S in liver tissues from wild-type and RASSF1A−/− littermates. Lysates were precipitated with Ac-K antibody and blotted with MAP1S-specific 4G1 antibody and Ac-K antibody. g Representative immunoblots showing the interaction of HDAC4 with RASSF1A in 293T cells transiently expressing Flag-HDAC4 and HA-RASSF1A. h, i Representative immunoblots (h) and quantification (i) showing the impact of RASSF1A depletion on levels of MAP1S, HDAC4, Bcl-2 and P27 in HeLa cells treated with random (Mock) or RASSF1A-specific siRNAs (RA). j, k Representative immunoblots (j) and quantification (k) showing the impact of overexpressed RASSF1A on levels of acetylated MAP1S in HeLa cells overexpressing HA-RASSF1A in the absence or presence of HDAC4 inhibitor apicidin (APCD). Lysates were precipitated with MAP1S-specific 4G1 antibody and blotted with Ac-K antibody and 4G1 antibody. l, m Representative immunoblots (l) and quantification (m) showing whether elevated levels of P27 affect the impact of RASSF1A on LC3-II levels in HeLa cells treated with random (Mock) or RASSF1A-specific siRNAs (RA) and transiently transfected with empty control vector or vector encoding P27 in the absence or presence of BAF
Fig 3: RASSF1A suppresses hepatocarcinogenesis and promotes survival in DEN-treated mice. a PCR analysis of DNA samples from mouse tails to genotype wild-type (RA+/+) and RASSF1A−/− mice (RA−/−). b Quantitative real-time PCR analysis of the levels of RASSF1A mRNA in liver tissues from wild-type and RASSF1A−/− mice. c Representative immunoblot showing levels of RASSF1A protein in hepatocytes isolated from wild-type and RASSF1A−/− mice. d Representative images of liver tissues from 12-month-old untreated wild-type and RASSF1A−/− mice in normal conditions. e Representative images of liver tissues from DEN-treated wild-type and RASSF1A−/− mice at different ages. f Plots of body weights, liver weights, ratios of body weight to liver weight of mice as shown in e. g Plots of number of surface tumors of mice as shown in e. h Comparative H&E staining among the liver tissues from DEN-treated 6-month-old mice described in e. Bar = 20 µm. i Plots of tumor size as shown in h. j Representative images showing different types of H&E staining of liver tissues from DEN-treated 12-month-old mice as shown in e. Bar = 20 µm. The percentage shown on top of each panel is the relative frequency of the type. k The Kaplan–Meier survival curves showing the survival times of male littermates of wild-type and RASSF1A−/− mice treated with DEN. n number of mice, MSD median survival days. The significance of difference between two groups was estimated by log-rank test and p value was the probability larger than the χ2 value. Here and later, all experiments were repeated at least three times. ns, not significant or p > 0.05; *p ≤ 0.05; **p ≤ 0.01; and ***p ≤ 0.001
Fig 4: MiR‐330‐3p mimics abrogated the function of RASSF1A knockdown in non‐small‐cell lung cancer (NSCLC) cell lines. A, Cell proliferation assay was evaluated by CCK‐8 assay. In A549 and NCI‐H23 cell lines, miR‐330‐3p inhibitor suppressed cell proliferation, whereas miR‐330‐3p mimics promoted it. Co‐transfection of mimics and RASSF1A inhibited this promotion. B,Transfection of NSCLC cell lines with miR‐330‐3p resulted in G0/G1 phase arrest by FACS flow cytometry. C, The apoptotic cells in the mimics group, inhibitor group and mimics + RASSF1A group were measured by flow cytometry. D, MiR‐330‐3p mimics increased cell invasion, which was reversed by RASSF1A. The data are from one representative experiment among three that were performed identically and are expressed as the means ± SD. *P < 0.05 and **P < 0.01
Fig 5: PDGFB is crucial for maintaining malignant properties induced by RASSF1A in NPC cells.a A heat map generated using the significantly changed genes categorized in the “cytokine-cytokine receptor interaction pathway” is shown. b, c mRNA expression (b) was evaluated by qRT-PCR and protein concentration by ELISA (c) in CM of RASSF1A-overexpressing CNE-2 cells, RASSF1A-depleted CNE-1 cells and their corresponding control cells, The data are presented as the mean ± S.D. values, **p < 0.01, Student’s t test. d–g PDGFB was transiently knocked down with a pool of siRNA or treated with a neutralizing antibody for PDGF-BB (10 µg/mL) in RASSF1A-depleted CNE-1 cells. PDGF-BB secretion in the CM was measured by ELISA (d), **p < 0.01, Student’s t test. e Number of spheroids formed was determined via microscopy, and representative images (e left panel) are shown. The formed spheroids were compared (e right panel), the data are presented as the mean ± S.D. values, *p < 0.05, **p < 0.01, Student’s t test; ns: non-sinificant. Scale bar: 200 µm. Representative images of the migration assay (f) and invasion assay (g) are shown, the data are presented as the mean ± S.D. values, **p < 0.01, Student’s t test. Scale bar: 100 µm. h–j Recombinant PDGF-BB or IgG was added to RASSF1A-overexpressing CNE-2 cells. Representative images of sphere formation (h) (Scale bar: 200 µm.), migration (i) and invasion (j) assays (Scale bar: 100 µm) of RASSF1A-overexpressing CNE-2 cells treated with PDGF-BB (the culture medium was supplemented with 20 ng/ml or an equal volume of control IgG) are shown, The data are presented as the mean ± S.D. values, *p < 0.05, **p < 0.01, Student’s t test.
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