Fig 2: Knockdown of clathrin HC impairs clathrin-mediated cargo uptake but does not affect Wnt signal transduction. (A) Clathrin and the AP2 complex are key components of the clathrin-mediated endocytic pathway. Clathrin HC (CLTC) and AP2 subunits a and µ were targeted. (B) Effective siRNA-mediated knockdown of CLTC was confirmed via Western blot. CLTC knockdown impaired cellular uptake of fluorescently labeled transferrin (p < 0.001). Scale bar, 20 µm. (C) Change in nuclear and (D) cellular ß-catenin upon stimulation with 5 nM Wnt3a was quantified in mESCs subjected to control or CLTC knockdown (control siRNA, n = 22; CLTC siRNA, n = 22; no Wnt3a, n = 15). (E) Transcription of the Wnt target gene Axin2 was assayed in mESCs subjected to control or CLTC knockdown. (F) Alkaline phosphatase stain intensity was quantified for individual mESC colonies in the specified culture condition (control siRNA MEKi+CHIR, n = 24; CLTC siRNA MEKi+CHIR, n = 12; control siRNA MEKi+Wnt3a, n = 29; CLTC siRNA MEKi+Wnt3a, n = 22; control siRNA MEKi, n = 23; CLTC siRNA MEKi, n = 21). (G) Cells in the bottom 20% of transferrin uptake function, as measured by low intracellular fluorescent transferrin signal, were sorted via FACS. Axin2 transcription was induced in these cells to a level similar to cells in the top 80% of transferrin uptake.

Fig 3: S pseudovirus transduces Huh 7 ?cells via clathrin-mediated endocytosis. A Huh 7 ?cells pretreated with different concentrations of camostat mesylate (5, 15, 50 µmol/L) or E-64d (5, 10, 25 ?µmol/L) were transduced with S pseudovirus and luciferase was measured. Values were normalized to control and expressed as mean ?± ?SD. B Huh 7 ?cells pretreated with lysosomotropic agents (50 ?mmol/L NH4Cl, 100 ?µmol/L chloroquine and 100 ?nmol/L bafilomycin A) were transduced with S pseudovirus or VSV-G pseudovirus. Luciferase was measured to indicate viral infection. Values were normalized to control and expressed as mean ?± ?SD. C Immunostaining of S pseudovirus infected Huh 7 ?cells for Spike (red) and EEA1 (green). Scalebar, 10 ?µm. D Huh 7 ?cells pretreated with indicated concentrations of pitstop were transduced with S pseudovirus or VSV-G pseudovirus and luciferase was measured. Values were normalized to control and expressed as mean ?± ?SD. E Huh 7 ?cells pretreated with indicated concentrations of CPZ were transduced with S pseudovirus or VSV-G pseudovirus and luciferase was measured. Values were normalized to control and expressed as mean ?± ?SD. F Huh 7 ?cells stably expressing a negative control or three independent CHC sgRNAs with Cas9 were transduced with S pseudovirus and luciferase was measured. Values were normalized to control and expressed as mean ?± ?SD. Inset, immunoblotting of CHC expression in Huh 7 cells as described above. S, spike; CQ, chloroquine; Baf A, bafilomycin A; CPZ, chlorpromazine; CHC, clathrin heavy chain; SD, standard deviation.

Fig 4: Clathrin-mediated endocytosis is not a significant modulator of Lrp6 or Frizzled receptor abundance on the plasma membrane. (A) FACS analysis of mESCs stained with OMP-18R5 and detected with Alexa-488 secondary showed that CLTC knockdown does not alter cell surface level of Frizzled receptors. (B) Immunoprecipitation of biotinylated cell surface proteins was followed by quantitative Western blot to detect surface Lrp6 with or without 30-min stimulation with 5 nM Wnt3a. The cytoplasmic control GAPDH was detected in lysates but not in immunoprecipitated cell surface fractions. (C) Immunoprecipitation of biotinylated cell surface proteins was followed by quantitative Western blot to detect surface Lrp6 with or without 8-h stimulation with 5 nM Wnt3a. (D) Quantification of surface Lrp6 signal normalized by total Lrp6 signal in Western blots in B and C.

Fig 5: Monodansylcadaverine blocks Wnt signaling independent of endocytosis. (A) Change in nuclear ß-catenin upon stimulation with 5 nM Wnt3a was quantified in mESCs in the presence of 150 µM MDC (Wnt3a, n = 33; Wnt3a with 150 µM MDC, n = 21; no Wnt3a, n = 30). (B) Wnt3a-induced transcription of Axin2 in mESCs and Wnt3a-induced TOPFlash luciferase activity in LS/L cells were assayed in the presence of increasing doses of MDC. (C) CHIR99021-induced transcription of Axin2 in mESCs and CHIR99021-induced TOPFlash luciferase activity in LS/L cells were assayed in the presence of increasing doses of MDC. (D) MDC blocked Wnt3a-induced Axin2 transcription in mESCs subjected to CLTC knockdown. (E) Western blot measured accumulation of ß-catenin in L cells stimulated with 5 nM Wnt3a for 2 h with increasing doses of MDC. Bar graph on the right shows ß-catenin signal normalized by GAPDH loading control signal averaged from two biological replicates.