Fig 2: R1 and R2 tubules differentially accommodate ER proteins. (A) Representative two-color STORM image of the immunolabeled endogenous Rtn4 (green) and calreticulin (magenta) in an untransfected COS-7 cell. (B and C) Representative two-color STORM images of the immunolabeled endogenous Rtn4 (green) and expressed ER-luminal protein mEmerald-ER-3 (magenta in B) and ER-membrane protein HaloTag-sec61b (magenta in C) in COS-7 cells. (D) Representative two-color STORM image of the immunolabeled endogenous Rtn4 (green) and calnexin (magenta) in an untransfected COS-7 cell. Filled and hollow arrowheads point to examples of R2 and R1 tubules, respectively. (Scale bars, 1 µm.)

Fig 3: Mechanisms of Nuclear Tunnels in Organizing Cellular Structures for Ribosome BiogenesisAll fluorescence images are Expansion Microscopy images taken on an Airyscan microscope with a 63x objective. All scale bars are in pre-expansion units.(A) i: Image of total lipid and total protein of a UCI082014 cell. Lipids were stained with mCling-atto647 (grey) and proteins were stained with Alexa Fluor 488 NHS ester (blue). Scale bar: 3 µm. ii: Magnified view of nuclear tunnel-nucleolus contact in the boxed area of the image (i). The arrow points to an FC/DFC region of the nucleolus. Scale bar: 500 nm. Length expansion factor: 3.9.(B) i: Image of an active nucleolus contacted by a nuclear tunnel in a UCI082014 cell. The cell was stained with anti-RPA194 antibodies (yellow), which mark Pol I, anti-Nup153 antibodies (green), which mark the nuclear tunnel, and NHS ester (grey). Scale bar: 5 µm. ii: Magnified view of NE-nucleolus contact in the boxed area of the image (i). Scale bar: 1 µm. Length expansion factor: 3.9.(C) i: Image of an inactive nucleolus without contact with NE in a UCI082014 cell. The cell was stained with anti-RPA194 antibodies (yellow), anti-Nup153 antibodies (green), and NHS ester (grey). Scale bar: 5 µm. ii: Magnified view of NE-nucleolus contact in the boxed area of the image (i). Scale bar: 1 µm. Length expansion factor: 3.9.(D) Barchart of RPA194 cluster density in the nucleoli contacted by tunnels or suspended in UCI082014 cells. Each bar represents the mean ± standard error of more than 15 nucleoli from 3 independent experiments. ** indicates p<0.01 by unpaired t test.(E) i: Image of an MCF-10A cell stained with anti-H3K9me3 antibodies (pink), anti-Nup153 antibodies (green), and NHS ester (grey). Scale bar: 3 µm. ii: Magnified view of tunnel-nucleolus contact in boxed area of image (i). Scale bar: 1 µm. Length expansion factor: 3.9.(F) i: Image of a UCI082014 cell stained with anti-eIF6 (orange) antibodies, anti-Nup153 (green), and NHS ester (grey). Scale bar: 3 µm. ii: Magnified view of tunnel-nucleolus contact in boxed area of image (i). Scale bar: 500 nm. Length expansion factor: 3.7.(G) i: Image of a UCI082014 cell stained with anti-Nup153 antibodies (green) and anti-exportin1 (magenta) antibodies. Scale bar: 5 µm. ii: Magnified view of a nuclear tunnel in boxed area of image (i). Scale bar: 500 nm. iii: Magnified view of Nup153 channel of image (ii). iv: Magnified view of exportin1 channel of image (ii). Length expansion factor: 4.0.(H) i: Image of a UCI082014 cell stained with anti-Sec61b antibodies (yellow) and anti-RPS3 (pink) antibodies, and NHS ester (blue). Scale bar: 5 µm. ii: Magnified view of tunnel-nucleolus contact in boxed area of image (i). Scale bar: 500 nm. Length expansion factor: 3.9.(I) i: Image of a UCI082014 cell stained with anti-Tom20 antibodies (purple) and NHS ester (blue) and. Scale bar: 5 µm. ii: Magnified view of tunnel-nucleolus contact in boxed area of image (i). Scale bar: 500 nm. Length expansion factor: 3.7.(J) i: Image of a UCI082014 cell stained with anti-alpha-tubulin antibodies (magenta) and NHS ester (blue). Scale bar: 3 µm. ii: Magnified view of tunnel-nucleolus contact in boxed area of image (i). Scale bar: 500 nm. Length expansion factor: 4.0.(K) Model of the organization of ribosome biogenesis-associated components near the nuclear tunnel

Fig 4: Identification of the nuclear localization of TfR1 and characterization of its nuclear translocation mechanism. a Representative IHC images (×10 magnification) of TfR1 staining in tumor tissues from different types of cancer (n = 204). Scale bar = 50 μm. b, c Fractionation of the nuclear and cytoplasmic components of HCT-116 cells in (b) and HCT-116 cells transfected with empty vector (EV) or Flag-TfR1 in (c). Lamin B1 was used as a nuclear (Nu) loading control. Tubulin was used as non-nuclear fraction (NNF), and whole cell lysate (WCL) loading control. d, e HCT-116 cells were treated with different concentrations of CPZ for 30 min in (d) or BFA for 6 h in (e) for immunoblot analysis of nuclear and cytoplasmic fractions. The relative abundance of nuclear TfR1 was normalized to that of the CPZ or BFA = 0 controls. Lamin B1 was used as a Nu loading control. Tubulin was used as the loading control for NNF and WCL. f Co-IP analysis of the interactions between Flag-TfR1 and Sec61β. GAPDH was used as a loading control. g HCT-116 cells were transfected with scrambled negative control siRNA (-) or SEC61B-targeted siRNA (+) for 72 h, followed by subcellular fractionation and immunoblot analysis. The relative abundance of nuclear TfR1 was normalized to that of the siRNA (-) control. Lamin B1 and Tubulin were used as equal loading controls for Nu, NNF, and WCL

Fig 5: land-ExM coupled with immunostaining LR-ExM for membrane-bound organelle visualization. (A–C) land-ExM total lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Tom20 antibodies (yellow). The anti-Tom20 antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–G) Magnified images of A–C showing details of mitochondria. Scale bar: 1 µm in pre-expansion unit. (H) Intensity profile along the cyan line across the mitochondria in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Sec61b antibodies (yellow). The anti-Sec61b antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (L–O) Magnified in images of I–K showing details of ER. Scale bar: 1 µm in pre-expansion unit. (P) Intensity profile along the cyan line across the ER in image (L). All images were taken with an Airyscan microscope.