Hart JE, Clarke, IJ, Risbridger GP, Ferneyhough, B,Vega-Hernández, M. Mysterious inhibitory cell regulator investigated and found likely

to be secretogranin II related.

Supplementary IHC Images

Mammalian and Drosophila IHC images indicating the tissue distribution of the endogenous antigen or antigens of the anti-EPL001 antibodies.

See article text for methodology and interpretation.

Figs. 1-30 are based on the use of the rabbit polyclonal anti-EPL001 antiserum ER88.

Figs. 31-38 involved the use of the goat polyclonal anti-EPL001 antiserum G530.

******

Key

‘micrin’ = The unknown mammalian antigen to the anti-EPL001 antisera in IHC is herein denoted thus by way of shorthand, implying no preconceptions as to chemical

identity. (Ref: Hart, JE. The body has a brake: micrin is a postulated new gonadal hormone curbing tissue overgrowth and restricting reproduction. Med Hypotheses.

2014; 83: 775-786.)

NE = Neuroendocrine

CgA = Chromagranin A

******

The thesis of the paper is that the observed anti-organotrophic hormonal activity is likely secretogranin II related. Fig. 1 is not evidential in this regard but is provided to apprise colleagues of the form in which the array tissues, human normal and tumour,

were supplied. Other tissues were sourced non-commercially.

Figure 1: Slides of human tissue array

Figure 2: Tissues - ve stained with micrin on the normal tissue array

Figure 3: Weakly stained with micrin on the normal tissue array

Figure 4: NE cells stained with micrin in prostate and gastrointestinal tract

Figure 5: Serial sections stained with micrin and CgA

Figure 6: Tissues +ve stained with micrin on the normal tissue array

Figure 7: Micrin staining with preabsorption on the human prostate samples

a & b x10c x d x40Antibody at 1/200PA = preabsorption with EPL001 peptide at 0.5 mg/ml

invasive folicular ca

Figure 9: Immunostaining micrin and CgA on human radical serial sections prostate

Figure 10 Normal Tumor

Figure 11 Normal Tumor

Figure 12

Figure 13 Normal Tumor

Figure 14

Pre

abso

rbed

Ant

ibod

y10

0µg/

ml

Nor

mal

Hum

an K

idne

y: IH

C

(ER

88 ra

bbit

poly

clon

al a

ntib

ody)

Ref

5. H

aylo

r JL,

Par

ker E

, Ris

brid

ger G

P, B

eale

D, B

row

n B

L, D

obso

n P

RM

, Cla

rke

IJ, H

art J

E. I

nhib

ition

of

com

pens

ator

y re

nal g

row

th b

y th

e N

-term

inus

of a

she

ep-d

eriv

ed p

eptid

e. R

egul

Pep

tides

. 200

9; 1

52: 4

8-53

.

Mag

nific

atio

n x1

00

Figure 15

. H

aylo

r JL,

Par

ker E

, Ris

brid

ger G

P, B

eale

D, B

row

n B

L, D

obso

n P

RM

, Cla

rke

IJ, H

art J

E. I

nhib

ition

of

No

diffe

renc

e se

en b

etw

een

ovar

y in

tact

and

ova

riect

omis

ed e

wes

in IH

C s

tain

ing

of h

ypot

hala

mic

med

ian

emin

ence

. N

ote:

‘Ter

min

al’ r

efer

s to

the

antib

ody

bein

g ra

ised

to th

e N

-term

inus

of a

pur

ified

ovi

ne p

rote

in

Figure 16

Imm

unoh

isto

chem

istry

for M

icrin

Rab

bit a

ntib

ody

used

at 1

/100

to 1

/100

0

DA

B v

isua

lisat

ion

No

stai

ning

with

pre

-imm

une

seru

m

Imm

unoc

hem

ical

ly st

aine

d ce

lls in

shee

p m

edia

n em

inen

ce (M

E),

shee

p ov

ary

(cor

pus l

uteu

m),

rat o

vary

(the

ca a

nd g

ranu

losa

) and

hu

man

pro

stat

e (b

asal

lum

inal

cel

ls)

Figure 17

Mic

rinin

ME:

1/1

000

x10

Figure 18

Mic

rinin

ME

1/10

00 x

40Figure 19

Mic

rinin

ME

1/10

00 x

40Figure 20

Mic

rin in

ME

x40

Mic

rin in

ME

x40

Figure 21

Mic

rin in

Rat

Ova

ry x

40Figure 22

Mic

rinin

shee

p ov

ary

1/10

00 x

10

Figure 23

Mic

rinin

shee

p ov

ary

1/10

00 x

40Figure 24

Mic

rin in

the

shee

p co

rpus

lute

um 1

/100

0 x1

0

Figure 25

Mic

rin in

the

shee

p co

rpus

lute

um 1

/100

0 x4

0

Figure 26

Hum

an p

rost

ate

BPH

= B

enig

n pr

osta

tic h

yper

plas

ia P

ca =

Pro

stat

ic c

arci

nom

a C

KH

MW

= p

an c

yto-

kera

tin (h

igh

mol

ecul

ar w

eigh

t), a

mar

ker o

f epi

thel

ial c

ells

in p

rost

ate

Figure 27

Mic

rince

lls s

tain

ed w

ith E

R88

in

she

ep P

VN

x10

X40

Figure 28

Mic

rinpo

sitiv

e th

eca

cells

inno

rmal

hum

an o

vary

X4X4

0

Figure 29

Mic

rinpo

sitiv

e ce

lls in

m

ouse

pla

cent

a

X4X4

0

Figure 30

Phot

omic

rogr

aph

of ra

t bra

in p

erfu

sed

with

4%

par

afor

mal

dehy

de a

nd se

ctio

ned

coro

nally

at 4

0 µm

. Sec

tions

blo

cked

with

nor

mal

ra

bbit

seru

m; p

rimar

y go

at a

ntib

ody

dilu

ted

at

1:10

00; i

ncub

atio

n at

4 d

egre

es C

for

48 h

ours

. Bio

tinyl

ated

rabb

it an

ti-go

at

seco

ndar

y (1

:500

for 1

hou

r) a

nd st

rep-

HR

P (1

:500

for 1

hou

r). C

olou

r dev

elop

ed u

sing

D

AB

(15m

in; R

oche

). A

rrow

poi

nts t

oim

mun

opos

itive

neu

rons

loca

ted

in re

gion

s of

the

hypo

thal

amus

. The

whi

te b

ay is

the

3rd

vent

ricle

(‘3V

’). S

cale

bar

50

µm

Figure 31

Figure

32.Epa

cooccurs

with

asubset

ofgliacells

intheDrosop

hilaventralcord

Pane

l(A)

show

saview

ofthe

ventralcordat

stage16

ona

wild

type

Drosop

hilaem

bryo

staine

dwith

antib

odiesa

gainst

endo

crineph

armaceu

tical’sgoat

antib

ody(Epa)(ingreen),Elav

(inred)

,and

Repo

(inblue

).Elav

isane

uralmarkere

xpressed

inthenu

cleu

sofallne

uralcells

.Re

poisexpressedinallglia.Epa

marks

themem

braneof

asub

seto

fcellsexpressin

gtheRe

ponu

clearm

arker.Pane

ls(B,B

1 ,B3

,B4)con

tainzoom

edinsertsfrom

(A)sho

wingallchann

elsmerge

andallind

ividualchann

els.(B

2)

show

scooccurringEp

aand

Repo

positivecells

(sho

wnin

insert).Althou

ghto

amuchless

extent

(B5 )show

sElav

andEp

aco

occurrance

(sho

wnin

insert).

(C1

C4)isa

digitalyre

arranged

zstackto

show

thecoronalview

of(A).(D

1D4)sho

wsa

lateral

view

of(A)after

digitalre

arrangem

ent.Ab

breviatio

nsV

(ven

tral),D(dorsal),

A/P

(anterior/po

sterior)

repo

epa

elav

A/P

V D

BB1

B2

B3B4

B5

C1 C2 C3 C4 D1 D2 D3 D4

AFigures 32

Figure

33.LateralView

(A))

show

salateralviewof

the

ventralcordat

stage16

onawild

type

Drosop

hilaem

bryo

staine

dwith

antib

odiesa

gainst

endo

crine

pharmaceu

tical’sgoat

antib

ody

(Epa)(ingreen),Elav(in

red)

,and

Repo

(inblue

).(B

B5)sho

ws

zoom

edinsertsfrom

(A)w

ithall

channe

lsmerge

andallind

ividual

channe

ls.Ab

breviatio

nsV

(ven

tral),D(dorsal),

A/P

(anterior/po

sterior

A BB1

B2

B3B4

B5

repo

epa

elav

A/P

Figures 33

Figure

34.Epa

antib

odywith

respecttoEagle

expressio

n.

(A)sho

wsa

view

ofthe

ventralcordat

stage

16on

aneg

kinesin

lacZ

Drosop

hilaem

bryo

staine

dwith

antib

odiesa

gainst

endo

crine

pharmaceu

tical’sgoat

antib

ody(Epa)(in

green),Elav(in

red)

,andlacZ

(inblue

).Eg

(eagle)isa

gene

involved

inde

term

inationof

serotone

rgicne

uron

s.(B)sho

wsa

higher

resolutio

npicturefrom

anarea

in(A).Pane

ls(B

B4)con

tain

digital

reslicing

of(B)w

here

white

dotted

lines

are

placed

.(C)

show

sexpressio

nof

Epa

with

intheDrosop

hila

brain(m

arkedinred

byElav

antib

ody).

Abbreviatio

nsV

(ven

tral),D(dorsal),

A/P

(anterior/po

sterior)

AB

B1 B2 B3 B4

C

eglacZ

epa

elav

V D

A/P

B1B2

B3 B4

Figures 34

AB

A1A2

A3A4

hb9

epa

eve

V DA/P

A5A6

B1B2

B3

B4B5

B6

A/P

Figure

35.Ep

aantib

odywith

respecttoEveandHb

9expressio

n(A)sho

wsa

view

oftheventralcordat

stage16

onawild

type

Drosop

hilaem

bryo

staine

dwith

antib

odiesa

gainst

endo

crineph

armaceu

tical’sgoat

antib

ody(Epa)(ingreen),Eve

(inred)

,and

Hb9(in

blue

).Pane

ls(A

1A6)con

sisto

fahigher

resolutio

nim

ageof

(A).

(B)

show

salateralviewof

theventralcordat

stage16

onawild

type

Drosop

hila.(BB6

)sho

ws

ahigher

resolutio

nim

ageof

(B).(B

5 )insertdisplayacoronalviewof

thedo

tted

area

toshow

thereisno

colocalizationof

themarkersAb

breviatio

nsV(ven

tral),D(dorsal),

A/P

(anterior/po

sterior)

Figures 35

V DA/P

heart

heart

gut

Figure

36.Ep

aantib

odywith

respecttomef2expressio

n(A)sho

wsa

view

oftheventralcordat

stage16

onawild

type

Drosop

hilaem

bryo

staine

dwith

antib

odiesa

gainst

endo

crine

pharmaceu

tical’sgoat

antib

ody(Epa)(ingreen)

andMef2(in

white).Mef2isatranscrip

tionfactor

impo

rtantd

uringmesod

erm

and

musclede

velopm

ent.Pane

ls(B)and

(D)con

sisto

ftwodiffe

rent

zplane

s ofthe

brainarea

(not

marked).A

bbreviations

V(ven

tral),

D(dorsal),

A/P(anterior/po

sterior)

AB

CD

epa

mef2

Figures 36

AB

CD

repo

EPA

elav

Fig.37

Diffe

rent

view

softhe

samedrosop

hilaem

bryo

brain.EPAingreen,elav

antib

ody

which

marks

allneu

ralcellsinredandrepo

antib

odymarking

theglialcellsinblue

AgainEPAco

localizes

with

repo

.A)isa

dorsalview

ofthebrain.B)

isamoreventralview.

C)Isadigitalreslicing

ofthesameim

ageseen

inAandBfrom

thelateralviewandD)

from

thetransverse

view

Figures 37

AB

CD

A’B’

C’D’

elav

epa

repo

merge

Fig.38

Controlexperim

entu

singEPL001

EPL001

Figures 38