Fig 2: 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 3: There are specific interactions among RASSF1A, MAP1S and LC3. a Representative immunoblot showing levels of Myc-LC3-II coimmunoprecipitated with HA-RASSF1A from lysates of MEFs transiently expressing HA-RASSF1A and Myc-LC3. b Representative immunoblot showing levels of GFP fused fragments of RASSF1A coimmunoprecipitated with LC3 from lysates of 293T cells transiently expressing GFP-LC3 and GFP fused RASSF1A constructs. c–e Representative immunoblots showing levels of RASSF1A coimmunoprecipitated with FL (c), HC (d), SC (e). f, g Representative immunoblots showing levels of LC (f) and HBD (g) coimmunoprecipitated with RASSF1A. h Representative immunoblots showing levels of RASSF1A coimmunoprecipitated with HBD in 293T cells transiently expressing HA-RASSF1A and HA-HBD. MAP1S-specific antibody 4G1 recognizes HBD. i Representative immunoblots showing levels of FL or FL∆ of MAP1S coimmunoprecipitated with RASSF1A in 293T cells transiently expressing HA-RASSF1A and HA-FL or HA-FL∆. j Representative immunoblots showing levels of RASSF1A constructs coimmunoprecipitated with endogenous MAP1S in 293T cells transiently expressing GFP-tagged RASSF1A constructs. k Representative immunoblots showing levels of RASSF1A or RA∆ coimmunoprecipitated with FL of MAP1S in 293T cells transiently expressing HA-FL and HA-RA or HA-RA∆. l Representative immunoblots showing the impact of MAP1S on levels of Myc-LC3-II coimmunoprecipitated with RASSF1A in wild-type and MAP1S−/− MEFs transiently expressing HA-RASSF1A and Myc-LC3-II. m Representative immunoblots showing levels of FL or FL∆ of MAP1S and Myc-LC3-II coimmunoprecipitated with RASSF1A in 293T cells transiently expressing HA-RASSF1A, Myc-LC3 and HA-fused FL or FLΔ

Fig 4: 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 5: RASSF1A suppresses PI3K-Akt-mTOR pathway to promote autophagy initiation through Hippo pathway regulatory protein MST1. a Representative immunoblots showing the fragment of RASSF1A coimmunoprecipitated with MST1 in 293T cells. b Representative immunoblots showing the impact of fragments of RASSF1A on the amount of MST1 coimmunoprecipitated with RASSF1A in 293T cells. Inputs are the same as in panel a. c, d Representative immunoblots (c) and quantification showing the impact of RASSF1A on levels of total and p-MST1 (d) in mouse liver tissues. e, f Representative immunoblots (e) and quantification (f) showing the impact of RASSF1A on levels of p-YAP, YAP, CTGF and Beclin 1 in mouse liver tissues. g, h Representative immunoblots showing levels of MST1 and Akt coimmunoprecipitated with RASSF1A (g) or levels of MST1 and RASSF1A coimmunoprecipitated with Akt (h) in HeLa cells. I, j Representative immunoblots (i) and quantification showing the impact of RASSF1A suppression on the levels of total MST1 or MST1 coimmunoprecipitated (IP-MST1) with Akt (j) in HeLa cells treated with random (Mock) or RASSF1A-specific siRNAs. k–r Representative immunoblots (k, m, o, q) and quantification showing the levels of p-AKT and p-S70S6K (l, n, p, r) in mouse liver tissues (k, l), MEFs (m, n), liver tissues from DEN-treated 6-month-old mice (o, p), or HeLa cells treated with random (Mock) or RASSF1A-specific siRNAs (q, r). s A diagram showing the mechanism by which RASSF1A promotes autophagy initiation through MST1-AKT-mTOR pathway