Fig 2: HYLS1 depletion interrupts the assembly of NPHP module at transition zone.RCTE cells transfected with negative control (siNC) or HYLS1-specific (siHYLS1-O1 and siHYLS1-O2) siRNAs for 48 hours were serum starved for 24 hours and then analyzed by IF microscopy. (A) TF components (CEP164, SCLT1, ODF2, and CEP83) were not affected by HYLS1 depletion. (B) IFT proteins IFT88 and IFT140 were not affected by HYLS1 depletion. (C) TZ components (MKS1, TCTN1, MKS5, and CEP290) were not affected by HYLS1 depletion. (D) NPHP1 and NPHP4, the core components of the NPHP module, were absent from the BB in HYLS1-depleted cells. PolyE-tub antibody labeled both the BB and the axoneme of cilia. Arl13b antibody labeled the ciliary shaft. Scale bars, 2 µm. The percentage of cells with normal localization of the tested protein was quantified in control or HYLS1-depleted cells (n > 100). Results from at least three independent experiments were statistically analyzed and plotted as means ± SEM. N.S., no significant difference. ***P < 0.001.

Fig 3: FBF1 controls adipose beiging through a cilia-specific PKA signaling(A and B) PKACa localization in WT and Fbf1tm/tm MEFs before and after treated with (A) CL-316,243 or (B) mirabegron. Centrosomes were stained with ?-tubulin. Scale bar, 1 µm.(C) Immunoblotting on vWAT or MEF extracts.(D) WT vWAT preadipocytes labeled with yellow fluorescent protein (YFP)-tagged CEP170C-PKI, CEP170C, and PKI. Centrosomes were stained with ?-tubulin. Scale bar, 1 µm.(E) WT vWAT preadipocytes overexpressed PKI, CEP170C, or CEP170C-PKI were treated with CL-316,243 and differentiated for 7 days in adipogenic medium (AM), then fixed, stained, and imaged. Green: lipids, blue: nuclei. Scale bar, 50 µm.(F) Relative mRNA levels of beiging genes in WT vWAT preadipocytes overexpressed PKI, CEP170C, or CEP170C-PKI.(G) Endogenous AKAP9 immunoprecipitates with FBF1 in 3T3-L1 cells.(H) AKAP9 localization in 3T3-L1 cells. FBF1 or ODF2 labeled basal bodies. Scale bar, 1 µm.(I) SIMS and 3-dimensional (3D) surface-rendering images of the basal bodies of 3T3-L1 cells stained for PKACa, AKAP9, and FBF1. Scale bar, 1 µm.(J) SIMS images of the basal bodies of 3T3-L1 cells stained for PKACa and FBF1. Scale bar, 1 µm.(K) Association between endogenous PKARI and FBF1 in 3T3-L1 cells treated with control or Akap9 siRNA.(L) 3T3-L1 cells treated with control or Akap9 siRNA were differentiated with CL-316,243 inductions for 7 days, then fixed, stained, and imaged. Green: lipids, blue: nuclei. Scale bar, 100 µm.(M) Relative mRNA levels of beiging genes in 3T3-L1 cells treated with control or Akap9 siRNA.

Fig 4: FBF1 controls Hh signaling by recruiting BBS chaperone complex to TFs(A) WT/Fbf1tm/tm MEFs were treated with DMSO or SAG and differentiated for 7 days, then fixed and stained with BIDIPY. Red: lipids, blue: nuclei. Scale bar, 150 µm.(B) Relative mRNA levels of adipogenic genes at day 4 of differentiation with or without AM in WT/Fbf1tm/tm MEFs treated with DMSO or SAG.(C–E) Localization of Smo (C), (D) Gli3, and (E) GPR161 in primary cilia of WT/Fbf1tm/tm MEFs treated with DMSO or SAG. Basal bodies and cilia were labeled with anti-?-tubulin and anti-acetylated tubulin. Scale bar, 5 µm.(F) Statistical analysis of Smo+, Gli3+, and GPR161+ cilia. Data were calculated from 300 cilia in each group. Values are expressed as means ± SEMs. ***p < 0.001.(G) Cilia length of WT and Fbf1tm/tm MEFs.(H) Gli1 mRNA level in WT and Fbf1tm/tm MEFs before and after Hh activation.(I) Localization of GLI3 in primary cilia of WT/Fbf1tm/tm vWAT tissues. Basal bodies and cilia were labeled with anti-?-tubulin and anti-acetylated tubulin. Scale bar, 5 µm.(J) Gli1 and Ptch1 mRNA levels in WT and Fbf1tm/tm vWAT tissues.(K) Gli1 mRNA level in WT and Fbf1tm/tm vWAT preadipocytes before and after Hh activation.(L) WT MEFs labeled with BBS12 and TF marker FBF1 or subdistal appendage marker ODF2, and analyzed with SIMS. Scale bar, 1 µm.(M) Endogenous BBS12 immunoprecipitates with FBF1 in 3T3-L1 cells.(N) Endogenous FBF1 immunoprecipitates with BBS12 in 3T3-L1 cells.(O) The localization of BBS12 in WT/Fbf1tm/tm MEFs. Anti-?-tubulin and anti-acetylated tubulin-labeled basal bodies and cilia. Scale bar, 5 µm.(P) Control or Bbs12-knockdown 3T3-L1 cells were overexpressed vehicle or FBF1 and differentiated for 7 days, then fixed, stained, and imaged. Red: lipids, blue: nuclei. Scale bar, 150 µm.(Q) SIM and 3D surface-rendering images of 3T3-L1 cells. Scale bar, 1 µm.(R) BBSome component BBS5 localization in WT/Fbf1tm/tm MEFs. Basal bodies were stained with ODF2. Scale bar, 1 µm.(S) BBSome component BBS4 localization in WT/Fbf1tm/tm MEFs. Basal bodies were stained with ODF2. Scale bar, 1 µm.

Fig 5: INPP5E inhibits the association of TTBK2 with the M-centriole/basal body.(a,b) TTBK2 was visualized by IF microscopy in serum-fed RCTE cells 72 h post transfection of the negative control (siNC), INPP5E-specific (siINPP5E) and/or PIPKI?-specific (siPIPKI?) siRNAs. ODF2 was used as a centrosome marker. (b) The centrosomal TTBK2 intensity in cells (n>30) of each group was quantified and plotted from three independent experiments. (c,d) RCTE cells treated with siNC or siINPP5E were collected for immunoblotting (c) or IF microscopy (d) with indicated antibodies. a-Tub, a-tubulin. (e,f) RCTE cells were transfected with empty vector (Control), wild type (WT) or catalytic defective (CD) Flag-INPP5E for 8 h, and serum starved for additional 8 h before stained with indicated antibodies. In each group, the intensity of centrosomal TTBK2 signal in >30 cells was quantified from at least three independent experiments and plotted. Error bars represent s.e.m. (g) Cells transfected with empty vector (Control), WT or CD Flag-INPP5E were collected for immunoblotting with indicated antibodies. (h–j) RCTE cells transfected with empty vector (Control) or wild-type Flag-INPP5E for 24 h were serum-starved for additional 36 h, and then stained with appropriate antibodies. Percentage of cells with CP110 on both centrioles (i; n>100) or with primary cilium (j; n>200) was quantified in each group. Results from at least three independent experiments were plotted. Error bars represent s.d. ***P<0.001. Scale bars, 0.5 µm.